Differential enzymatic fragmentation

ABSTRACT

The present invention provides methods for detecting the presence of methylation at a locus within a population of nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. patent application Ser. No.10/971,986, filed Oct. 21, 2004 and claims the benefit of U.S. PatentApplication Nos. 60/513,426, filed Oct. 21, 2003, 60/561,721, filedApr., 12, 2004, and 60/561,563, filed Apr. 12, 2004, the disclosures ofeach of which are hereby incorporated by reference in their entirety forall purposes.

BACKGROUND OF THE INVENTION

DNA typically comprises both methylated and unmethylated bases.Prokaryotic DNA is methylated at cytosine and adenosine residues (see,e.g., McClelland et al., Nuc. Acids. Res. 22:3640-3659 (1994).Methylation of prokaryotic DNA protects the DNA from digestion bycognate restriction enzymes, i.e., foreign DNAs (which are notmethylated in this manner) that are introduced into the cell aredegraded by restriction enzymes which cannot degrade the methylatedprokaryotic DNA. DNA methylation patterns can be used to identifyspecific bacterial types (e.g., genus, species, strains, and isolates)

Mammalian DNA can only be methylated at cytosine residues, typicallythese cytosines are 5′ neighbors of guanine (CpG). This methylation hasbeen shown by several lines of evidence to play a role in gene activity,cell differentiation, tumorigenesis, X-chromosome inactivation, genomicimprinting and other major biological processes (Razin and Riggs eds. inDNA Methylation Biochemistry and Biological Significance,Springer-Verlag, N.Y., 1984).

In eukaryotic cells, methylation of cytosine residues that areimmediately 5′ to a guanosine, occurs predominantly in CG poor loci(Bird, Nature 321:209 (1986)). In contrast, discrete regions of CGdinucleotides called CpG islands remain unmethylated in normal cells,except during X-chromosome inactivation and parental specific imprinting(Li, et al., Nature 366:362 (1993)) where methylation of 5′ regulatoryregions can lead to transcriptional repression.

Aberrant methylation, including aberrant methylation at specific loci,is often associated with a disease state. For example, de novomethylation of the Rb gene has been demonstrated in a small fraction ofretinoblastomas (Sakai, et al., Am. J. Hum. Genet., 48:880 (1991)), anda more detailed analysis of the VHL gene showed aberrant methylation ina subset of sporadic renal cell carcinomas (Herman, et al., PNAS USA,91:9700 (1994)). Expression of a tumor suppressor gene can also beabolished by de novo DNA methylation of a normally unmethylated 5′ CpGisland. See, e.g., Issa, et al., Nature Genet. 7:536 (1994); Merlo, etal., Nature Med. 1:686 (1995); Herman, et al., Cancer Res., 56:722(1996); Graff, et al., Cancer Res., 55:5195 (1995); Herman, et al.,Cancer Res. 55:4525 (1995). Methylation of the p16 locus is associatedwith pancreatic cancer. See, e.g., Schutte et al., Cancer Res.57:3126-3131 (1997). Methylation changes at the insulin-like growthfactor II/H19 locus in kidney are associated with Wilms tumorigenesis.See, e.g., Okamoto et al., PNAS USA 94:5367-5371 (1997). The associationof alteration of methylation in the p15, E-cadherin and vonHippel-Lindau loci are also associated with cancers. See, e.g., Hermanet al., PNAS USA 93:9821-9826 (1997). The methylation state of GSTP1 isassociated with prostate cancer. See, e.g., U.S. Pat. No. 5,552,277.Therefore, detection of altered methylation profiles at loci where suchalterations are associated with disease can be used to provide diagnosesor prognoses of disease.

Current methods for determining whether DNA is methylated orunmethylated typically used methylation-sensitive restriction enzymes ora combination of methylation-sensitive and methylation-insensitiverestriction enzymes (see, e.g., Burman et al., Am. J. Hum. Genet.65:1375-1386 (1999); Toyota et al., Cancer Res. 59:2307-2312 (1999);Frigola et al., Nucleic Acids Res. 30(7):e28 (2002); Steigerwald et al.,Nucleic Acids Res. 18(6):1435-1439 (1990); WO 03/038120; and U.S. patentPublication No. 2003/0129602 A1). In these methods, methylated DNAsequences remain intact for analysis.

Thus, there is a need in the art for more efficient methods of detectingmethylation of DNA, particularly DNA at specific loci. The presentinvention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of detecting methylation at alocus within a population of nucleic acids using a methylation-dependentrestriction enzyme, a methylation sensitive restriction enzyme, and/or amethylation-insensitive restriction enzyme.

One embodiment of the invention provides a method of detecting thepresence of methylation at a locus within a population of nucleic acidsby (a) dividing the population of nucleic acids into at least twoportions, (b) contacting a first portion with a methylation-sensitiverestriction enzyme to obtain a population comprising fragmentedunmethylated copies of the locus and intact methylated copies of thelocus; (c) quantifying the intact copies of the locus in the firstportion; (d) contacting a second portion with a methylation-dependentrestriction enzyme to obtain a population comprising fragmentedmethylated copies of the locus and intact unmethylated copies of thelocus; (e) quantifying the intact copies of the locus in the secondportion; and (f) determining the presence of methylation at the locus bycomparing the number of intact copies of the locus in the first portionand number of intact copies of the locus in the second portion. In someembodiments, the method further comprises quantifying a third portion ofthe nucleic acids, thereby amplifying the total intact copies of thelocus in the population; and comparing the number of total intact copiesof the locus to the number of intact copies of the locus in the firstportion and/or intact copies of the locus in the second portion. In someembodiments, the method further comprises contacting a third portion ofthe nucleic acids with a methylation-dependent restriction enzyme and amethylation-sensitive restriction enzyme; quantifying copies of theintact locus in the third portion; and determining the presence ofmethylation at the locus by comparing the number of the intact copies ofthe locus in the third portion to the number of intact copies of thelocus in the first portion and/or intact unmethylated copies of thelocus in the second portion. In some embodiments the second portion isalso contacted with the methylation-sensitive restriction enzyme priorto the step of quantifying the intact copies of the locus in the secondportion. In some embodiments, the first portion is also contacted withthe methylation-dependent restriction enzyme prior to the step ofquantifying the intact copies of the locus in the first portion. In someembodiments, the method further comprises contacting a fourth portion ofthe nucleic acids with a methylation-dependent restriction enzyme and amethylation-sensitive restriction enzyme; quantifying intact copies ofthe locus in the fourth portion; and determining the presence ofmethylation at the locus by comparing the number of intact copies of thelocus in the fourth portion to the number of total intact copies of thelocus in the third portion and/or intact copies of the locus in thefirst portion and/or intact copies of the locus in the second portion.In some embodiments, the method further comprises identification ofmutations within the locus. In one embodiment, the method furthercomprises contacting a fifth portion of the nucleic acids with amethylation-insensitive restriction enzyme; quantifying intact copies ofthe locus in the fifth portion to obtain a population of nucleic acidscomprising a mutation at the locus; and determining the presence of amutation at the locus by comparing the number of the intact copies ofthe locus in the fifth portion to the number of intact copies of thelocus in the fourth portion and/or total intact copies of the locus inthe third portion and/or intact copies of the locus in the first portionand/or intact copies of the locus in the second portion.

In some embodiments, the number of unmethylated copies of the locus isdetermined by subtracting the number of intact copies of the locusremaining after the first portion is cut with the methylation-sensitiverestriction enzyme from the total intact copies of the locus. In someembodiments, the number of methylated copies of the locus is determinedby subtracting the number of intact copies of the locus remaining afterthe second portion is cut with the methylation-dependent restrictionenzyme from the total intact copies of the locus. In some embodiments,the number of hemimethylated and mutant copies of the locus isdetermined by (a) subtracting the number of intact copies of the locusremaining after the first portion is cut with the methylation-sensitiverestriction enzyme from the total intact copies of the locus, therebydetermining the number of unmethylated copies of the locus; and (b)subtracting the number of unmethylated copies of the locus from thenumber of intact copies of the locus remaining after the second portionis cut with the methylation-dependent restriction enzyme, therebydetermining the number of hemimethylated and mutant copies of the locus.In some embodiments, the number of hemimethylated and mutant copies ofthe locus is determined by: (a) subtracting the number of intact copiesof the locus remaining after the second portion is cut with themethylation-dependent restriction enzyme from the total intact copies ofthe locus, thereby determining the number of methylated copies of thelocus; and (b) subtracting the number of methylated copies of the locusfrom the number of intact copies of the locus remaining after the firstportion is cut with the methylation-sensitive restriction enzyme,thereby determining the number of hemimethylated and mutant copies ofthe locus. In some embodiments the number of methylated and unmethylatedcopies of the locus is determined by subtracting the number of intactcopies of the locus remaining after the fourth portion is cut with themethylation-dependent restriction enzyme and the methylation-sensitiverestriction enzyme from the total intact copies of the locus, therebydetermining the number of methylated and unmethylated copies of thelocus. In some embodiments, the number of hemimethylated copies of thelocus is determined by subtracting the number of intact loci remainingafter the fifth portion of the nucleic acids is contacted with amethylation-insensitive restriction enzyme from the number of intactcopies of the locus remaining after the fourth portion is cut with themethylation-dependent restriction enzyme and the methylation-sensitiverestriction enzyme. In some embodiments, the number of methylated copiesof the locus is determined by (a) subtracting the number of intactcopies of the locus remaining after the fifth portion of the nucleicacids is contacted with a methylation-insensitive restriction enzymefrom the number of intact copies of the locus remaining after the fourthportion is cut with the methylation-dependent restriction enzyme and themethylation-sensitive restriction enzyme, thereby determining the numberof hemimethylated copies of the locus; and (b) subtracting the number ofhemimethylated copies of the locus and the number of intact copies ofthe locus remaining after the fifth portion of the nucleic acids iscontacted with the methylation-insensitive restriction enzyme from thenumber of intact copies of the locus remaining after the first portionof nucleic acids is contacted with the methylation-sensitive restrictionenzyme, thereby determining the number of methylated copies of thelocus. In some embodiments, the number of unmethylated copies of thelocus is determined by (a) subtracting the number of intact copies ofthe locus remaining after the fifth portion of the nucleic acids iscontacted with a methylation-insensitive restriction enzyme from thenumber of intact copies of the locus remaining after the fourth portionis cut with the methylation-dependent restriction enzyme and themethylation-sensitive restriction enzyme, thereby determining the numberof hemimethylated copies of the locus; and (b) subtracting the number ofhemimethylated copies of the locus and the number of intact copies ofthe locus remaining after the fifth portion of the nucleic acids iscontacted with the methylation-insensitive restriction enzyme from thenumber of intact copies of the locus remaining after the second portionof nucleic acids is contacted with the methylation-dependent restrictionenzyme, thereby determining the number of unmethylated copies of thelocus.

In some embodiments, the quantifying steps comprise the direct detectionof intact copies of locus with hybrid capture. In some embodiments, thequantifying steps comprise quantitative amplification including, e.g.,quantitative PCR. In some embodiments, the quantitative amplificationproduct is detected by detecting a label intercalated between bases ofdouble stranded DNA sequences. In some embodiments, the quantitativeamplification product is detected by detecting hybridization of adetectably labeled oligonucleotide to the amplification product. In someembodiments, the detectably labeled oligonucleotide is a labeledoligonucleotide probe comprising a fluorophore and a hairpin structureor a dual labeled oligonucleotide probe comprising a pair of interactivelabels (e.g., a quencher and a fluorophore). In some embodiments, thefluorophore is activated to generate a detectable signal when the probehybridizes to its target nucleic acid sequence. In some embodiments, thefluorophore is activated to generate a detectable signal when the probehybridizes to its target nucleic acid sequence and an enzyme with 5′exonuclease activity cleaves the portion of the probe comprising thequencher.

In some embodiments, the nucleic acids are contacted with an agent thatmodifies unmethylated cytosines before the amplification step; and theintact copies of the locus are amplified with a pair of oligonucleotideprimers comprising at least one primer that distinguishes betweenmodified methylated and unmethylated DNA.

In some embodiments, the nucleic acids are contacted with an agent(e.g., sodium bisulfite) that modifies unmethylated cytosines before theamplification step; and the intact copies of the locus are amplifiedwith a pair of oligonucleotide primers comprising at least one primerthat distinguishes between the protected methylated and the modifiedunmethylated DNA.

In some embodiments, the methylation-dependent restriction enzyme iscontacted to the second, third, or fourth portion under conditions thatallow for at least some copies of potential restriction enzyme cleavagesites for the methylation-dependent restriction enzyme in the locus toremain uncleaved. In some embodiments, the density of methylation at thelocus is determined by comparing the number of intact methylated loci inthe second, third, or fourth portion after cleavage with a control valuerepresenting the quantity of methylation in a control DNA.

In some embodiments, the methylation-sensitive restriction enzyme iscontacted to the first or third portion under conditions that allow forat least some copies of potential restriction enzyme cleavage sites forthe methylation-sensitive restriction enzyme in the locus to remainuncleaved. In some embodiments, the density of methylation at the locusis determined by comparing the number of intact unmethylated loci in thefirst or third portion after cleavage with a control value representingthe quantity of methylation in a control DNA.

In some embodiments, the methylation is at the C4 position of acytosine, the C5 position of a cytosine within the locus, or at the N6position of an adenosine within the locus.

In some embodiments, the nucleic acids are DNA, including, e.g., genomicDNA.

In some embodiments, the methylation-sensitive restriction enzyme doesnot cut when a cytosine within the recognition sequence is methylated atposition C5.

In some embodiments, the methylation-sensitive restriction enzyme is AatII, Aci I, Acl I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I, BsaA I,BsaH I, BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I,Cla I, Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinC II, Hpa II,Hpy99 I, HpyCH4 IV, Kas I, Mbo I, Mlu I, MapA1 I, Msp I, Nae I, Nar I,Not I, Pml I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I, SfoI, SgrA I, Sma I, SnaB I, Tsc I, Xma I, and Zra I.

In some embodiments, the methylation-sensitive restriction enzyme doesnot cut when an adenosine within the recognition sequence is methylatedat position N6.

In some embodiments, the methylation-sensitive restriction enzyme is MboI.

In some embodiments, the methylation-dependent restriction enzyme isMcrBC, McrA, MrrA, or Dpn I.

In some embodiments, the first portion of genomic nucleic acids isfurther contacted with at least a second methylation-sensitiverestriction enzyme.

In some embodiments, the second portion of genomic nucleic acids isfurther contacted with at least a second methylation-dependentrestriction enzyme.

In some embodiments, the methylation-sensitive restriction enzyme ismethyl-adenosine sensitive. In some embodiments, themethylation-sensitive restriction enzyme is methyl-cytosine sensitive.

In some embodiments, the methods further comprise contacting a thirdportion of the nucleic acids with a second methylation-sensitiverestriction enzyme and contacting a fourth portion of the nucleic acidswith a second methylation-dependent restriction enzyme; quantifyingintact loci in the third and fourth portions; and determining thepresence of methylation at the locus by comparing the number of intactcopies of the locus in the third and fourth portions to the number oftotal intact copies of the locus and/or intact methylated copies of thelocus and/or intact unmethylated copies of the locus. In someembodiments, the first methylation-sensitive restriction enzyme ismethyl-cytosine sensitive; the second methylation-sensitive restrictionenzyme is methyl-adenosine sensitive; the first methylation-dependentrestriction enzyme is methyl-cytosine sensitive; and the secondmethylation dependent enzyme is methyl-adenosine sensitive.

In some embodiments, the presence of methylation at the locus iscompared between at least two nucleic acid samples (e.g., isolated fromat least two organisms having the same phenotype or a differentphenotype). In some embodiments, the methods comprise quantifying theintact methylated copies of the locus in a first sample and a secondsample; and comparing the quantity of amplified products from the twosamples, thereby determining relative methylation at the locus betweenthe two samples. In some embodiments, a first nucleic acid sample isisolated from a cell suspected of being a cancer cell and a secondnucleic acid sample is isolated from a non-cancerous cell.

In some embodiments, the presence of methylation at two or more loci isdetermined by quantifying copies of intact DNA at the two different locifrom a first portion contacted with a methylation-dependent restrictionenzyme; quantifying the intact DNA as the two loci from a second portioncontacted with a methylation-sensitive restriction enzyme; and comparingthe quantities of the amplified products for the two loci.

In some embodiments, the population of nucleic acids is isolated from acell, including, e.g., a plant cell, a fungal cell, a prokaryotic cell,an animal cell, a mammalian cell, or a cancer cell.

In some embodiments, the population of nucleic acids is isolated from asample from a subject (e.g., a human), including, e.g., a body fluid, asecretion, or a tissue biopsy. In some embodiments, the subject issuspected of having cancer.

In some embodiments, the locus comprises a sequence that is moremethylated in diseased cells than in non-diseased cells. In someembodiments, the locus comprises a sequence that is less methylated indiseased cells than in non-diseased cells.

In some embodiments, the methods further comprise adding sequence tagsonto the ends of a population of nucleic acids before dividing thepopulation of nucleic acids into equal portions, and step (c) comprisesquantifying the remaining intact methylated copies of the locus in thefirst portion with primers that initiate amplification from the sequencetags; and step (e) comprises quantifying the remaining unmethylatedintact copies of the locus in the second portion with primers thatinitiate amplification from the sequence tags.

These and other embodiments of the invention are further illustrated bythe detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates how the methods of the invention can be used todetermine methylation at a locus.

FIG. 2 illustrates results of amplification of DNA at differentmethylated:unmethylated dilutions.

FIG. 3 illustrates the ability of McrBC to distinguish between DNA atdifferent methylated:unmethylated dilutions. The arrows at the bottom ofthe figure indicate the approximate ΔCt between HhaI-cut and HhaI/McrBCdouble cut samples.

FIG. 4 illustrates analysis of DNA at a 1:2000 methylated:unmethylateddilution.

FIG. 5 illustrates a plot of change in cycle threshold as a function ofdilution of methylated/unmethylated DNA.

FIG. 6 illustrates results from different methylated:unmethylateddilutions.

FIG. 7 illustrates a hypothetical methylation density progression in thedevelopment of disease.

FIG. 8 illustrates McrBC DNA restriction.

FIG. 9 illustrates amplification results from different McrBC dilutionsrestricting sparsely-methylated DNA.

FIG. 10 illustrates amplification results from different McrBC dilutionsrestricting densely-methylated DNA.

FIG. 11 illustrates using different restriction enzyme dilutions todetermine optimum resolution between DNA with different methylationdensities.

FIG. 12 illustrates what data is obtained when the methylation state ofonly particular nucleotides is detected in a hypothetical diseaseprogression.

FIG. 13 illustrates what data is obtained when the average methylationdensity of a locus is detected in a hypothetical disease progression.

FIG. 14 depicts a portion of the p16 promoter (SEQ ID NO:1) methylatedin vitro with M.Sss I.

FIG. 15 illustrates data demonstrating that methylation-dependent (i.e.,McrBC) and methylation-sensitive (i.e., Aci I) restriction enzymesdistinguish different methylation densities at a DNA locus.

FIG. 16 illustrates cycle threshold data demonstrating thatmethylation-dependent (i.e., McrBC) and methylation-sensitive (i.e., AciI) restriction enzymes distinguish different methylation densities at aDNA locus.

FIG. 17 illustrates a consensus restriction map of kafirin genes. Therelevant restriction sites are indicated vertically and the numbersindicate the distances scale in base-pairs. Each coding sequence isdepicted as the blue-shaded arrow, and the region assayed is indicatedby the black bar. The circles depict sites that are not present in everykafirin gene, and the color represents the number of genes that do notshare the site. The orange circle (5′ most HhaI site) is conserved in 9of 11 Kafirin genes, and the red circle (3′ most PstI site) is presentin 10 of the 11.

FIG. 18 illustrates the heterogenous CG methylation and homogenous CNGmethylation of eleven kafirin genes.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides methods of detecting the presence ofmethylation at a locus within a population of nucleic acids usingmethylation-sensitive restriction enzymes, methylation-dependentrestriction enzymes, and/or methylation-insensitive enzymes. Accordingto the methods of the invention, nucleic acids comprising the locus ofinterest are cut with one or more restriction enzymes as describedherein to generate populations of intact methylated loci, intactunmethylated loci, and/or intact hemimethylated loci. The loci areamplified, and the amplification products are compared. Comparison ofthe amplification products provides information regarding methylation atthe locus.

DNA methylation plays important roles in gene regulation and thereforeit is desirable to evaluate genomic methylation for numerous purposes.For example, the presence or absence of methylation at a particularlocus can provide diagnostic and prognostic information regardingdiseases associated with aberrant methylation at the locus.

II. Definitions

“Methylation” refers to cytosine methylation at C5 or N4 positions ofcytosine (“5mC” and “4mC,” respectively), at the N6 position of adenine(“6mA”), or other types of nucleic acid methylation. Aberrantmethylation of a DNA sequence (i.e., hypermethylation orhypomethylation) may be associated with a disease, condition orphenotype (e.g., cancer, vascular disease, cognitive disorders, or otherepigenetic trait). An “unmethylated” DNA sequence contains substantiallyno methylated residues at least at recognition sequences for aparticular methylation-dependent or methylation-sensitive restrictionenzyme used to evaluate the DNA. “Methylated” DNA contains methylatedresidues at least at the recognition sequences for a particularmethylation-dependent or methylation-sensitive restriction enzyme usedto evaluate the DNA. It is understood that while a DNA sequence referredto as “unmethylated” may generally have substantially no methylatednucleotides along its entire length, the definition encompasses nucleicacid sequences that have methylated nucleotides at positions other thanthe recognition sequences for restriction enzymes. Likewise, it isunderstood that while a DNA sequence referred to as “methylated” maygenerally have methylated nucleotides along its entire length, thedefinition encompasses nucleic acid sequences that have unmethylatednucleotides at positions other than the recognition sequences forrestriction enzymes. “Hemimethylated” DNA refers to double stranded DNAin which one strand of DNA is methylated at a particular locus and theother strand is unmethylated at that particular locus.

A “methylation-dependent restriction enzyme” refers to a restrictionenzyme that cleaves at or near a methylated recognition sequence, butdoes not cleave at or near the same sequence when the recognitionsequence is methylated. Methylation-dependent restriction enzymesinclude those that cut at a methylated recognition sequence (e.g., DpnI)and enzymes that cut at a sequence that is at the recognition sequence(e.g., McrBC). For example, McrBC require two half-sites. Each half-sitemust be a purine followed by 5-methyl-cytosine (R5mC) and the twohalf-sites must be no closer than 20 base pairs and no farther than 4000base pairs away from each other (N20-4000). McrBC generally cuts closeto one half-site or the other, but cleavage positions are typicallydistributed over several base pairs approximately 32 base pairs from themethylated base. Exemplary methylation-dependent restriction enzymesinclude, e.g., McrBC (see, e.g., U.S. Pat. No. 5,405,760), McrA, MrrA,and Dpn I. One of skill in the art will appreciate that homologs andorthologs Of the restriction enzymes described herein are also suitablefor use in the present invention.

A “methylation-sensitive restriction enzyme” refers to a restrictionenzyme (e.g., PstI) that cleaves at or in proximity to an unmethylatedrecognition sequence but does not cleave at or in proximity to the samesequence when the recognition sequence is methylated. Exemplarymethylation sensitive restriction enzymes are described in, e.g.,McClelland et al, Nucleic Acids Res. 22(17):3640-59 (1994) and//rebase.neb.com. Suitable methylation-sensitive restriction enzymesthat do not cleave at or near their recognition sequence when a cytosinewithin the recognition sequence is methylated at position C⁵ include,e.g., Aat II, Aci I, Ad I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I,BsaA I, BsaH I, BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I, BstN I,BstU I, Cla I, Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinC II, HpaII, Hpy99 I, HpyCH4 IV, Kas I, Mbo I, Mlu I, MapA1 I, Msp I, Nae I, NarI, Not I, Pml I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I,Sfo I, SgrA I, Sma I, SnaB I, Tsc I, Xma I, and Zra I. Suitablemethylation-sensitive restriction enzymes that do not cleave at or neartheir recognition sequence when an adenosine within the recognitionsequence is methylated at position N⁶ include, e.g., Mbo I. One of skillin the art will appreciate that homologs and orthologs of therestriction enzymes described herein are also suitable for use in thepresent invention. One of skill in the art will further appreciate thata methylation-sensitive restriction enzyme that fails to cut in thepresence of methylation of a cytosine at or near its recognitionsequence, may be insensitive to the presence of methylation of anadenosine at or near its recognition sequence. Likewise, amethylation-sensitive restriction enzyme that fails to cut in thepresence of methylation of an adenosine at or near its recognitionsequence, may be insensitive to the presence of methylation of acytosine at or near its recognition sequence. For example, Sau3AI issensitive (i.e., fails to cut) to the presence of a methylated cytosineat or near its recognition sequence, but is insensitive (i.e., cuts) tothe presence of a methylated adenosine at or near its recognitionsequence.

A “methylation insensitive restriction enzyme” refers to a restrictionenzyme that cuts DNA regardless of the methylation state of the base ofinterest (A or C) at or near the recognition sequence of the enzyme. Oneof skill in the art will appreciate that a methylation-insensitiverestriction enzyme that cuts in the presence of methylation of anadenosine at or near its recognition sequence, may be sensitive to thepresence of methylation of a cytosine at or near its recognitionsequence, i.e., will fail to cut. Likewise, a methylation-insensitiverestriction enzyme that cuts in the presence of methylation of acytosine at or near its recognition sequence, may be sensitive to thepresence of methylation of an adenosine at or near its recognitionsequence. For example, Sau3AI is insensitive (i.e., cuts) to thepresence of a methylated adenosine at or near its recognition sequence,but is sensitive (i.e., fails to cut) to the presence of a methylatedcytosine at or near its recognition sequence.

“Isoschizomers” refer to distinct restriction enzymes have the samerecognition sequence. As used in this definition, the “same recognitionsequence” is not intended to differentiate between methylated andunmethylated sequences. Thus, an “isoschizomeric partner” of amethylation-dependent or methylation-sensitive restriction enzyme is arestriction enzyme that recognizes the same recognition sequence as themethylation-dependent or methylation-sensitive restriction enzymeregardless of whether a nucleotide in the recognition sequence ismethylated.

As used herein, a “recognition sequence” refers to a primary nucleicacid sequence and does not reflect the methylation status of thesequence.

The “methylation density” refers to the number of methylated residues ina given locus of DNA divided by the total number of nucleotides in thesame DNA sequence that are capable of being methylated. Methylationdensity is determined for cytosines only or adenosines only.

“Dividing” or “divided” in the context of dividing DNA, typically refersto dividing a population of nucleic acids isolated from a sample intotwo or more physically distinct portions, each of which comprise all ofthe sequences present in the sample. In some cases, “dividing” or“divided” refers to dividing a population of nucleic acids isolated froma sample into two or more physically distinct parts that do not containall of the sequences present in the sample.

Cleaving DNA “under conditions that allow for at least some copies ofpotential restriction enzyme cleavage sites in the locus to remainuncleaved” refers to any combination of reaction conditions, restrictionenzyme and enzyme concentration and/or DNA resulting in at least some ofthe DNA comprising a potential restriction enzyme cleavage site toremain uncut. For example, a partial digestion of the DNA (e.g., bylimiting the amount of enzyme or the amount of time of the digestion)allows some potential restriction enzyme cleavage sites in the locus toremain uncut. Alternatively, a complete digestion using a restrictionenzyme such as McrBC will result in some potential restriction enzymecleavage sites in the locus to remain uncut because the enzyme does notalways cut between the two recognition half sites, thereby leaving atleast some uncleaved copies of a locus in a population of sequenceswherein the locus is defined by the two recognition half-sites. A“potential restriction enzyme cleavage site” refers to a sequence that arestriction enzyme is capable of cleaving (i.e., comprising theappropriate nucleotide sequence and methylation status) when itrecognizes the enzymes recognition sequence, which may be the same ordifferent from the cleavage site.

A “partial digestion” of DNA as used herein refers to contacting DNAwith a restriction enzyme under appropriate reaction conditions suchthat the restriction enzyme cleaves some (e.g., less than about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) but not all of possiblecleavage sites for that particular restriction enzyme in the DNA. Apartial digestion of the sequence can be achieved, e.g., by contactingDNA with an active restriction enzyme for a shorter period of time thanis necessary to achieve a complete digestion and then terminating thereaction, by contacting DNA with less active restriction enzyme than isnecessary to achieve complete digestion with a set time period (e.g.,30, 60, 90, 120, 150, 150, or 240 minutes), or under other alteredreaction conditions that allow for the desired amount of partialdigestion. “Possible sites” are generally enzyme recognition sequences,but also include situations where an enzyme cleaves at a sequence otherthan the recognition sequence (e.g., McrBC).

A “complete digestion” of DNA as used herein refers to contacting DNAwith a restriction enzyme for sufficient time and under appropriateconditions to allow for cleavage of at least 95%, and typically at least99%, or all of the restriction enzyme recognition sequences for theparticular restriction enzyme. Conditions, including the time, buffersand other reagents necessary for complete digestions are typicallyprovided by manufacturers of restriction enzymes. Those of skill in theart will recognize that the quality of the DNA sample may preventcomplete digestion.

“An agent that modifies unmethylated cytosine” refers to any agent thatalters the chemical composition of unmethylated cytosine but does notchange the chemical composition of methylated cytosine. Examples of suchagents include sodium bisulfite, sodium metabisulfite, permanganate, andhydrazine.

“Genomic DNA” as used herein refers to the entire genomic sequence, or apart thereof, of an individual. A “subset” of the genomic DNA refers toa part of the entire genomic sequence, i.e., a subset contains onlysome, but not all of the loci of an entire genome. The individual may bean animal, a plant, a fungus, or a prokaryote, as well as a cell,tissue, organ, or other part of an organism.

A “methylation profile” refers to a set of data representing themethylation state of DNA from e.g., the genome of an individual or cellsand tissues of an individual. The profile can indicate the methylationstate of every base pair in an individual or can comprise informationregarding a subset of the base pairs (e.g., the methylation state ofspecific restriction enzyme recognition sequence) in a genome.

“Amplifying” DNA refers to any chemical, including enzymatic, reactionthat results in an increased number of copies of a template nucleic acidsequence or an increased signal indicating that the template nucleicacid is present in the sample. Amplification reactions includepolymerase chain reaction (PCR) and ligase chain reaction (LCR) (seeU.S. Pat. Nos. 4,683,195 and 4,683,202; PCR PROTOCOLS: A GUIDE TOMETHODS AND APPLICATIONS (Innis et al., eds, 1990)), strand displacementamplification (SDA) (Walker, et al. Nucleic Acids Res. 20(7):1691-6(1992); Walker PCR Methods Appl 3(1):1-6 (1993) transcription-mediatedamplification (Phyffer, et al., J. Clin. Microbiol. 34:834-841 (1996);Vuorinen, et al., J. Clin. Microbiol. 33:1856-1859 (1995)), nucleic acidsequence-based amplification (NASBA) (Compton, Nature 350(6313):91-2(1991), rolling circle amplification (RCA) (Lisby, Mol. Biotechnol.12(1):75-99 (1999)); Hatch et al., Genet. Anal. 15(2):35-40 (1999));branched DNA signal amplification (bDNA) (see, e.g., Iqbal et al., Mol.Cell Probes 13(4):315-320 (1999)), and linear amplification. Amplifyingincludes, e.g., ligating adaptors that comprise T3 or T7 promoter sitesto the template nucleic acid sequence and using T3 or T7 polymerases toamplify the template nucleic acid sequence.

A “locus” as used herein refers to a target sequence within a populationof nucleic acids (e.g., a genome). If a single copy of the targetsequence is present in the genome, then “locus” will refer to a singlelocus. If multiple copies of the target sequence are present in thegenome, then “locus” will refer to all loci that contain the targetsequence in the genome.

III. Detecting Methylation at a Locus within a Nucleic Acid Population

Methods of the invention may comprise comparing the presence or absenceor amounts of intact DNA following restriction of a sample divided intoat least two portions, wherein the portions are treated with differentrestriction enzymes. In many embodiments, a first portion is contactedwith a methylation-dependent restriction enzyme (producing intactunmethylated DNA and fragmented methylated DNA) and a second portion iscontacted with a methylation-sensitive restriction enzyme (producingintact methylated DNA and fragmented unmethylated DNA). The intactcopies of the locus from each portion are analyzed after the restrictiondigests and compared.

In some embodiments, a third portion of nucleic acids comprising thelocus is not digested with a restriction enzyme to provide an analysisof the total number of intact copies of a locus in a sample. The totalnumber of the intact copies of the locus can be compared to the numberof methylated loci and/or the number of unmethylated loci to verify thatthe number of methylated loci and unmethylated loci are equal to thetotal number of loci.

In further embodiments, a fourth portion of nucleic acids comprising thelocus is digested with both the methylation-sensitive restriction enzymeand the methylation-dependent restriction enzyme and any intact loci arequantified (e.g., quantitatively amplified or detected by hybridcapture). The total number of intact loci remaining after the doubledigestion can be compared to the number of methylated copies of thelocus, unmethylated copies of the locus, and/or total copies of thelocus to verify that the number of methylated copies and unmethylatedcopies are equal to the total number of copies and to verify that thecutting of the methylation sensitive and methylation dependentrestriction enzymes is complete.

In even further embodiments, a fifth portion of nucleic acids comprisingthe locus is digested with a methylation-insensitive restriction enzyme(i.e., either insensitive to methylation of an adenosine or of acytosine residue at its recognition sequence) and any intact copies ofthe locus are detected. The total number of intact copies remainingafter digestion can be compared to the number of methylated copies,unmethylated copies, and/or total copies to verify that the cutting ofthe other methylation sensitive and methylation dependent restrictionenzymes is complete; and/or to identify mutations in copies of the locusthat affect the recognition site of the methylation sensitive andmethylation dependent restriction enzymes.

Thus, a comparison of at least five separate amplified nucleic acidpopulations can be made:

-   -   (1) an untreated or mock treated population where virtually all        of the copies of the locus remain in tact;    -   (2) a population treated with a methylation-dependent        restriction enzyme where virtually all of the unmethylated        copies of the locus remain intact;    -   (3) a population treated with a methylation-sensitive        restriction enzyme where virtually all of the methylated copies        of the locus remain intact;    -   (4) a population treated with both a methylation-dependent        restriction enzyme and a methylation-sensitive restriction        enzyme which contains no or few intact copies of the locus; and    -   (5) a population treated with a methylation insensitive        restriction enzyme (i.e., either insensitive to methylation at        an adenosine or at a cytosine residue at or near its recognition        sequence) which contains no or few intact copies of the locus,        except copies of the locus that are mutated at the recognition        site of the restriction enzyme.

Typically, the samples are divided into equal portions, each of whichcontains all of the sequences present in the sample. In some cases, thesamples may be divided into parts that do not contain all of thesequences present in the sample. However, by comparing results fromdifferent combinations of restriction digests, the number of methylatedand unmethylated copies of the locus of interest can be determined. Anyof the above populations can thus be compared to any other population.For example, populations (1) and (2) can be compared with one another;or either population (1) or (2) can be compared with another population,e.g., population (4).

The order in which the digest(s) are performed are not critical. Thus,although it may be preferable to perform a digest in a certain order,e.g., to first digest with a particular class of methylation-sensingenzymes, e.g., methylation-sensitive enzymes, it is not necessary.Similarly, it may be preferable to perform a double digest.

In some embodiments, the nucleic acid may be obtained from a samplecomprising a mixed population of members having different methylationprofiles. For example, a biological sample may comprise at least onecell type with little or no methylation at a locus of interest and atleast one cell type that is methylated at the locus. The proportion ofthe population constituting methylated or unmethylated loci can beassessed by determining the amount of undigested loci in asingle-digested aliquot treated with only methylation-sensitive ormethylation-dependent restriction enzyme(s) to the amount of undigestedDNA in an aliquot treated with both methylation-sensitive andmethylation-dependent restriction enzymes. As used in this context, a“single” digest may, in practice, be performed using more than oneenzyme that is methylation-sensitive, or more than one enzyme that ismethylation-dependent, whether used sequentially or simultaneously. Forexample, an aliquot that is digested with more than onemethylation-sensitive restriction enzyme, but no methylation-dependentrestriction enzymes is considered a “single” digest. A “double” digestis considered to be an aliquot that has been treated using bothmethylation-sensitive and methylation-dependent restriction enzymes,whether used sequentially or simultaneously, regardless of the number ofmethylation-sensitive and methylation-dependent restriction enzymesemployed.

The amount of undigested DNA in the single digest relative to the doubledigest and the total number of copies of the locus in the sample isindicative of the proportion of cells that contain unmethylated vsmethylated DNA at the locus of interest. Furthermore, such an analysiscan serve as a control for the efficacy of the single digest, e.g. thepresence of a detectable change in the amount of undigested DNA in thedouble digest compared to the amount in the single digest with amethylation-sensitive restriction enzyme is an indication that thesingle digest went to completion.

One of skill in the art will appreciate that, by selecting appropriatecombinations of restriction enzymes (e.g., methylation-sensitive,methylation-dependent, and methylation-insensitive restriction enzymes),the methods of the invention can be used to determine cytosinemethylation or adenosine methylation at a particular locus based on,e.g., the recognition sequence of the restriction enzyme. For example,by cutting a first portion of nucleic acids comprising a locus ofinterest with a methylation-sensitive restriction enzyme which fails tocut when a methylated cytosine residue is in its recognition sequence(e.g., Hha I), and cutting a second portion of nucleic acids comprisinga locus of interest with a methylation-dependent restriction enzymewhich cuts only if its recognition sequence comprises a methylatedcytosine (e.g., McrBC), the cytosine methylation of a particular locusmay be determined. Likewise, by cutting a first portion of nucleic acidscomprising a locus of interest with a methylation-sensitive restrictionenzyme which fails to cut when an adenosine residue is methylated in itsrecognition sequence (e.g., Mbo I), and cutting a second portion ofnucleic acids comprising a locus of interest with amethylation-dependent restriction enzyme which cuts in the presence ofmethylated adenosines in its recognition sequence (e.g., Dpn I), theadenosine methylation of a particular locus may be determined. In someembodiments, all four sets of digestions are conducted in parallel forboth adenosine methylation and cytosine methylation to simultaneouslydetermine the adenosine methylation and the cytosine methylation of aparticular locus.

In addition, restriction enzymes that are either sensitive to methylatedcytosine or to methylated adenosine can be used in the methods of theinvention to provide populations of cytosine methylated loci andadenosine methylated loci for comparison.

Suitable methylation-dependent restriction enzymes include, e.g., McrBC,McrA, MrrA, and DpnI. Suitable methylation-sensitive restriction enzymesinclude restriction enzymes that do not cut when a cytosine within therecognition sequence is methylated at position C⁵ such as, e.g., Aat II,Aci I, Acl I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I, BsaA I, BsaH I,BsiE I, BsiW L, BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I, Cla I,Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinC II, Hpa II, Hpy99 I,HpyCH4 IV, Kas I, Mlu I, MapA1 I, Msp I, Nae I, Nar I, Not I, Pml I, PstI, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I, Sfo I, SgrA I, Sma I,SnaB I, Tsc I, Xma I, and Zra I. Suitable methylation-sensitiverestriction enzymes include restriction enzymes that do not cut when anadenosine within the recognition sequence is methylated at position N⁶such as, e.g., Mbo I. One of skill in the art will appreciate thathomologs and orthologs of the restriction enzymes described herein arealso suitable for use in the present invention.

In some embodiments, the nucleic acid portions are treated with an agentthat modifies a particular unmethylated base, such as sodium bisulfite,prior to treatment with restriction enzymes. The nucleic acids can thenbe treated and amplified using at least one primer that distinguishesbetween protected methylated and modified unmethylated nucleotides. Theamplified portions are then compared to determine relative methylation.Certain quantitative amplification technologies employ one or moredetection probes that are distinct from the amplification primers. Thesedetection probes can also be designed to discriminate between protectedmethylated and modified unmethylated DNA.

This invention relies on routine techniques in the field of recombinantgenetics. For example, methods of isolating genomic DNA, digesting DNAwith restriction enzymes, ligating oligonucleotide sequences, detectingamplified and unamplified DNA, and sequencing nucleic acids are wellknown in the art. Basic texts disclosing the general methods of use inthis invention include Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL (3rd ed. 2001); Kriegler, GENE TRANSFER AND EXPRESSION: ALABORATORY MANUAL (1990); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(Ausubel et al., eds., 2001)).

A. Digestion with Restriction Enzymes

Either partial or complete restriction enzyme digestions, depending onthe restriction enzyme, can be used to provide information regarding themethylation density within a particular DNA locus. The restrictionenzymes for use in the invention are typically selected based on asequence analysis of the locus of interest. One or more enzymes in eachcategory (e.g., methylation-dependent or methylation-sensitive) are thenselected. The sequence analysis can be performed based on evaluatingdatabases of known sequences or in some instances, can be based onempirical determinations, e.g., to take into account variants such asmutations, that may be present in a particular subject.

1. DNA Samples

DNA can be obtained from any biological sample can be used, e.g., fromcells, tissues, and/or fluids from an organism (e.g., an animal, plant,fungus, prokaryote). The samples may be fresh, frozen, preserved infixative (e.g., alcohol, formaldehyde, paraffin, or PreServeCyte™) ordiluted in a buffer. Biological samples include, e.g., skin, blood or afraction thereof, tissues, biopsies (from e.g., lung, colon, breast,prostate, cervix, liver, kidney, brain, stomach, esophagus, uterus,testicle, skin, bone, kidney, heart, gall bladder, bladder, and thelike), body fluids and secretions (e.g., blood, urine, mucus, sputum,saliva, cervical smear specimens, marrow, feces, sweat, condensedbreath, and the like). Biological samples also include, leaves, stems,roots, seeds, petals, pollen, spore, mushroom caps, and sap.

2. Complete Digestion

When a DNA sample comprising a locus of interest is completely digestedwith a methylation sensing restriction enzyme (i.e., amethylation-dependent or methylation sensitive restriction enzyme), theinformation provided includes the presence or absence of methylation atrecognition sequences of the restriction enzyme. The presence of intactDNA in a locus comprising the cut site of the restriction enzymeindicates that the appropriate methylation state of the recognition sitenecessary for cleavage by the methylation-sensitive ormethylation-dependent restriction enzyme was not present at or near thelocus.

The amount of intact DNA can be compared to a control representing anequal amount of DNA from the sample that was not contacted with therestriction enzyme. Alternatively, the amount of intact DNA at a locuscan be compared to a second locus or to the same locus in DNA isolatedfrom another cell. In another alternative, the amount of intact DNA at alocus can be compared to DNA having a known or expected number ofmethylated and monitorable restriction sites. In some embodiments, theDNA being compared is approximately the same size. Those of skill in theart will appreciate that other controls are also possible. Thus, bydetecting the amount of intact DNA at the locus following restrictionenzyme digestion, the relative number of methylated alleles isdetermined.

Use of restriction enzymes that have a variable cleavage pattern nearthe recognition sequence (e.g., McrBC) provides a special case forcomplete digestions of DNA. In this case, even if the locus contains arecognition sequence in the appropriate methylation state, some of thefragments containing a methylated locus will remain intact becausecleavage of the DNA will occur outside the locus according to a functionof probability. Therefore, a complete digestion with McrBC behavessimilarly to a partial digestion with a methylation sensitiverestriction enzyme (which cuts at its recognition site) with respect tothe number of intact alleles.

The mechanism of McrBC DNA cutting occurs as follows. An eight subunitcomplex of McrB binds to each of two recognition half sites(purine-methylC represented as (A or G)mC). See FIG. 8. These complexesthen recruit one McrC subunit to their respective half sites and startto translocate along the DNA mediated by GTP hydrolysis. When two McrBCbound complexes contact each other, a double-complex is formed andrestriction occurs. Cutting will generally not occur if the two halfsites are closer than 20 bp and restriction resulting from half sites asfar as 4 kb from one another have been observed, though are rare.Restriction may occur ˜32 bp to the left or right of either bound halfsite, giving four possible cut site locations: ˜32 bp 5′ of the firsthalf site, ˜32 bp 3′ of the first half site, ˜32 bp 5′ of the secondhalf site, and ˜32 bp 3′ of the second half site. Therefore, it ispossible for two half sites to exist within a locus defined by PCRprimers and for cleavage to occur outside of the locus. It is alsopossible for two half sites to exist outside of the locus and for a cutto occur within the locus. It is also possible for one site to exist inthe locus and for another to exist outside of the locus and for a cut tooccur either within or outside of the locus. Thus, the more methylatedhalf sites that are “in the vicinity” of the locus (whether literallybetween the amplification primers or in neighboring flanking sequence),the more likely a cut will be observed within the locus for a givenconcentration of McrBC. Accordingly, the number of copies of amethylated locus that are cleaved by McrBC in a complete or partialdigestion will be proportional to the density of methylated nucleotides.

3. Partial Digestions

The amount of cleavage with a methylation sensitive ormethylation-dependent restriction enzyme in a partial (i.e., incomplete)digestion, reflects not only the number of fragments that contain anyDNA methylation at a locus, but also the average methylation densitywithin the locus of DNA in the sample. For instance, when DNA fragmentscontaining the locus have a higher methylation density, then a partialdigestion using a methylation-dependent restriction enzyme will cleavethese fragments more frequently within the locus. Similarly, when DNAfragments containing the locus have a lower methylation density, then apartial digestion using a methylation-dependent restriction enzyme willcleave these fragments less frequently within the locus, because fewerrecognition sites are present. Alternatively, when a methylationsensitive restriction enzyme is used, DNA fragments with a highermethylated density are cleaved less, and thus more intact DNA strandscontaining the locus are present. In each of these cases, the digestionof DNA sample in question is compared to a control value such as thosediscussed above for complete digestions.

In some embodiments, the DNA sample can be split into equal portions,wherein each portion is submitted to a different amount of partialdigestion with McrBC or another methylation-dependent restrictionenzyme. The amount of intact locus in the various portions (e.g., asmeasured by quantitative DNA amplification) can be compared to a controlpopulation (either from the same sample representing uncut DNA orequivalent portions from another DNA sample). In cases where theequivalent portions are from a second DNA sample, the second sample canhave an expected or known number of methylated nucleotides (or at leastmethylated restriction enzyme recognition sequences) or, alternatively,the number of methylated recognition sequences can be unknown. In thelatter case, the control sample will often be from a sample ofbiological relevance, e.g., from a diseased or normal tissue, etc.

In some embodiments, the DNA sample is partially digested with one ormore methylation-sensitive restriction enzymes and then amplified toidentify intact loci. Controls in these cases are similar to those usedfor methylation-dependent restriction enzyme digestions described above.Untreated controls are undigested, and any treated control DNA samplesare digested with methylation-sensitive restriction enzymes.

It can be useful to test a variety of conditions (e.g., time ofrestriction, enzyme concentration, different buffers or other conditionsthat affect restriction) to identify the optimum set of conditions toresolve subtle or gross differences in methylation density among two ormore samples. The conditions may be determined for each sample analyzedor may be determined initially and then the same conditions may beapplied to a number of different samples.

4. Generation of Control Values

Control values can represent either external values (e.g., the number ofintact loci in a second DNA sample with a known or expected number ofmethylated nucleotides or methylated restriction enzyme recognitionsequences) or internal values (e.g., a second locus in the same DNAsample or the same locus in a second DNA sample). While helpful, it isnot necessary to know how many nucleotides (i.e. the absolute value) inthe control are methylated. For example, for loci in which methylationresults in a disease state, knowledge that the locus is more methylatedthan it is in normal cells can indicate that the subject from which thesample was obtained may have the disease or be in the early stages ofdeveloping disease.

In cases where the same DNA sample includes a control locus, multiplexamplification, e.g., multiplex PCR, can be used to analyze two more loci(e.g., at least one target locus and at least one control locus).

DNA samples can vary by two parameters with respect to methylation: (i)the percentage of total copies in a population that have any methylationat a specific locus, and (ii) for copies with any DNA methylation, theaverage methylation density among the copies. It is ideal, though notrequired, to use control DNAs that evaluate both of these parameters ina test sample.

Control DNAs with known methylated cytosines are produced using anynumber of DNA methylases, each of which can have a different targetmethylation recognition sequence. This procedure can create a populationof DNA fragments that vary with respect to the methylation density(i.e., the number of methylated cytosines per allele). Partial methylasereactions can also be used, e.g., to produce a normally distributedpopulation with a mode at the average methylation density for thepopulation. In some embodiments, the mode can be adjusted for a givenpopulation as a function of the completeness of the methylase reaction.Control DNAs can also be synthesized with methylated and unmethylatedDNA bases.

In some cases, a DNA target with a known sequence is used. A desiredcontrol DNA can be produced by selecting the best combination ofmethylases and restriction enzymes for the analysis. First, a map ofsites that can be methylated by each available methylase is generated.Second, a restriction map of the locus is also produced. Third,methylases are selected and are used to in vitro methylate the controlDNA sample to bring about a desired methylation pattern, which isdesigned to perform optimally in combination with the restrictionenzymes used in the methylation analysis of the test DNA and control DNAsamples. For example, M.HhaI methylates the site (G*CGC) and McrBCrecognizes two half sites with the motif (RpC). Therefore, eachmethylated M.HhaI site in the control sequence is recognized by McrBC.

Similarly, a population of molecules may be then treated with a DNAmethylase (e.g., M.SssI) in the presence of magnesium to result in adesired methylation density. If the reaction is allowed to run tocompletion, nearly all of the sites that can be methylated will bemethylated, resulting in a high and homogeneous methylation density. Ifthe reaction is limited in its course, a lower average methylationdensity (or partial methylation) will result (i.e., all possible sitesare not methylated due to timing of reaction and/or concentration ofenzyme). In this way, the desired average methylation density of thecontrol DNA can be produced. The methylated control DNA can be preciselycharacterized by determining the number of methylated cytosines throughbisulfite sequencing. Alternatively, the methylation control DNA can beprecisely characterized by determining the number of methylatedcytosines through a comparison to other known control DNAs as describedherein.

For more precise prediction of methylation densities, it may be usefulto generate a control set of DNA that can conveniently serve as astandard curve where each sample in the control set has a differentmethylation density, either known or unknown. By cutting the multiplesamples with a methylation-dependent restriction enzyme or amethylation-sensitive restriction enzyme under conditions that allow forat least some copies of potential restriction enzyme cleavage sites inthe locus to remain uncleaved and subsequently amplifying the remainingintact copies of a locus, a standard curve of the amount of intactcopies (e.g., represented by Ct values) can be generated, therebycorrelating the amount of intact DNA to different methylation densities.The standard curve can then be used to determine the methylation densityof a test DNA sample by interpolating the amount of intact DNA in thesample following restriction and amplification as described herein.

B. Calculation of Methylation Density Based on Cycle Thresholds

As described herein, cycle thresholds (Ct) are a useful measurement fordetermining the initial amount of DNA template in an amplificationreaction. Accordingly, Ct values from samples treated with amethylation-dependent and/or methylation-sensitive restriction enzymeand amplified as described herein can be used to calculate methylationdensity. A change in Ct value between one sample and a control value(which can represent the Ct value from a second sample) is predictive ofrelative methylation density. Because amplification in PCR theoreticallydoubles copies every cycle, 2^(X) is proportional to the number ofcopies in the amplification during exponential amplification, where X isthe number of cycles. Thus 2^(Ct) is proportional to the amount ofintact DNA at the initiation of amplification. The change of Ct (ΔCt)between two samples or between a sample and a control value (e.g.,representing a Ct value from a control) represents the difference ininitial starting template in the samples. Therefore, 2^(|ΔCt|) isproportional to the relative methylation density difference between asample and a control or a second sample. For instance, a difference of1.46 in the Ct between two samples (each treated with amethylation-dependent restriction enzyme and subsequently amplified)indicates that one sample has approximately 2.75 (i.e., 2^((1.46))=2.75)times more methylated nucleotides within the monitored sites at a locusthan the other sample.

C. Detecting DNA

The presence and quantity of DNA cleaved by the restriction enzymes canbe detected using any means known in the art including, e.g.,quantitative amplification, hybrid capture, or combinations thereof.

1. Quantitative Amplification

In some embodiments, the presence and quantity of DNA cleaved by therestriction enzymes can be determined by amplifying the locus followingdigestion. By using amplification techniques (e.g., the polymerase chainreaction (PCR)) that require the presence of an intact DNA strand foramplification, the presence and amount of remaining uncut DNA can bedetermined. For example, PCR reactions can be designed in which theamplification primers flank a particular locus of interest.Amplification occurs when the locus comprising the two primers remainsintact following a restriction digestion. If the amount of total andintact DNA is known, the amount of cleaved DNA can be determined. Sincecleavage of the DNA depends on the methylation state of the DNA, theintact and cleaved DNA represents different methylation states.

Amplification of a DNA locus using reactions is well known (see U.S.Pat. Nos. 4,683,195 and 4,683,202; PCR PROTOCOLS: A GUIDE TO METHODS ANDAPPLICATIONS (Innis et al., eds, 1990)). Typically, PCR is used toamplify DNA templates. However, alternative methods of amplificationhave been described and can also be employed, as long as the alternativemethods amplify intact DNA to a greater extent than the methods amplifycleaved DNA.

DNA amplified by the methods of the invention can be further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence such as PCR, oligomer restriction(Saiki, et al., Bio/Technology 3:1008-1012 (1985)), allele-specificoligonucleotide (ASO) probe analysis (Conner, et al., PNAS USA 80:278(1983)), oligonucleotide ligation assays (OLAs) (Landegren, et al.,Science 241:1077, (1988)), and the like. Molecular techniques for DNAanalysis have been reviewed (Landegren, et al., Science 242:229-237(1988)).

Quantitative amplification methods (e.g., quantitative PCR orquantitative linear amplification) can be used to quantify the amount ofintact DNA within a locus flanked by amplification primers followingrestriction digestion. Methods of quantitative amplification aredisclosed in, e.g., U.S. Pat. Nos. 6,180,349; 6,033,854; and 5,972,602,as well as in, e.g., Gibson et al., Genome Research 6:995-1001 (1996);DeGraves, et al., Biotechniques 34(1):106-10, 112-5 (2003); Deiman B, etal., Mol Biotechnol. 20(2):163-79 (2002). Amplifications may bemonitored in “real time.”

In general, quantitative amplification is based on the monitoring of thesignal (e.g., fluorescence of a probe) representing copies of thetemplate in cycles of an amplification (e.g., PCR) reaction. In theinitial cycles of the PCR, a very low signal is observed because thequantity of the amplicon formed does not support a measurable signaloutput from the assay. After the initial cycles, as the amount of formedamplicon increases, the signal intensity increases to a measurable leveland reaches a plateau in later cycles when the PCR enters into anon-logarithmic phase. Through a plot of the signal intensity versus thecycle number, the specific cycle at which a measurable signal isobtained from the PCR reaction can be deduced and used to back-calculatethe quantity of the target before the start of the PCR. The number ofthe specific cycles that is determined by this method is typicallyreferred to as the cycle threshold (Ct). Exemplary methods are describedin, e.g., Heid et al. Genome Methods 6:986-94 (1996) with reference tohydrolysis probes.

One method for detection of amplification products is the 5′-3′exonuclease “hydrolysis” PCR assay (also referred to as the TaqMan™assay) (U.S. Pat. Nos. 5,210,015 and 5,487,972; Holland et al., PNAS USA88: 7276-7280 (1991); Lee et al., Nucleic Acids Res. 21: 3761-3766(1993)). This assay detects the accumulation of a specific PCR productby hybridization and cleavage of a doubly labeled fluorogenic probe (the“TaqMan™” probe) during the amplification reaction. The fluorogenicprobe consists of an oligonucleotide labeled with both a fluorescentreporter dye and a quencher dye. During PCR, this probe is cleaved bythe 5′-exonuclease activity of DNA polymerase if, and only if, ithybridizes to the segment being amplified. Cleavage of the probegenerates an increase in the fluorescence intensity of the reporter dye.

Another method of detecting amplification products that relies on theuse of energy transfer is the “beacon probe” method described by Tyagiand Kramer, Nature Biotech. 14:303-309 (1996), which is also the subjectof U.S. Pat. Nos. 5,119,801 and 5,312,728. This method employsoligonucleotide hybridization probes that can form hairpin structures.On one end of the hybridization probe (either the 5′ or 3′ end), thereis a donor fluorophore, and on the other end, an acceptor moiety. In thecase of the Tyagi and Kramer method, this acceptor moiety is a quencher,that is, the acceptor absorbs energy released by the donor, but thendoes not itself fluoresce. Thus, when the beacon is in the openconformation, the fluorescence of the donor fluorophore is detectable,whereas when the beacon is in hairpin (closed) conformation, thefluorescence of the donor fluorophore is quenched. When employed in PCR,the molecular beacon probe, which hybridizes to one of the strands ofthe PCR product, is in the open conformation and fluorescence isdetected, while those that remain unhybridized will not fluoresce (Tyagiand Kramer, Nature Biotechnol. 14: 303-306 (1996)). As a result, theamount of fluorescence will increase as the amount of PCR productincreases, and thus may be used as a measure of the progress of the PCR.Those of skill in the art will recognize that other methods ofquantitative amplification are also available.

Various other techniques for performing quantitative amplification of anucleic acids are also known. For example, some methodologies employ oneor more probe oligonucleotides that are structured such that a change influorescence is generated when the oligonucleotide(s) is hybridized to atarget nucleic acid. For example, one such method involves is a dualfluorophore approach that exploits fluorescence resonance energytransfer (FRET), e.g., LightCycler™ hybridization probes, where twooligo probes anneal to the amplicon. The oligonucleotides are designedto hybridize in a head-to-tail orientation with the fluorophoresseparated at a distance that is compatible with efficient energytransfer. Other examples of labeled oligonucleotides that are structuredto emit a signal when bound to a nucleic acid or incorporated into anextension product include: Scorpions™ probes (e.g., Whitcombe et al.,Nature Biotechnology 17:804-807, 1999, and U.S. Pat. No. 6,326,145),Sunrise™ (or Amplifluor™) probes (e.g., Nazarenko et al., Nuc. AcidsRes. 25:2516-2521, 1997, and U.S. Pat. No. 6,117,635), and probes thatform a secondary structure that results in reduced signal without aquencher and that emits increased signal when hybridized to a target(e.g., Lux probes™).

In other embodiments, intercalating agents that produce a signal whenintercalated in double stranded DNA may be used. Exemplary agentsinclude SYBR GREEN™ and SYBR GOLD™. Since these agents are nottemplate-specific, it is assumed that the signal is generated based ontemplate-specific amplification. This can be confirmed by monitoringsignal as a function of temperature because melting point of templatesequences will generally be much higher than, for example,primer-dimers, etc.

2. Methylation State-Specific Amplification

In some embodiments, methylation-specific PCR can be employed to monitorthe methylation state of specific nucleotides in a DNA locus. In theseembodiments, following or preceding digestion with the restrictionenzyme, the DNA is combined with an agent that modifies unmethylatedcytosines. For example, sodium bisulfite is added to the DNA, therebyconverting unmethylated cytosines to uracil, leaving the methylatedcytosines intact. One or more primers are designed to distinguishbetween the methylated and unmethylated sequences that have been treatedwith sodium bisulfite. For example, primers complementary to thebisulfite-treated methylated sequence will contain guanosines, which arecomplementary to endogenous cytosines. Primers complementary to thebisulfite-treated unmethylated sequence will contain adenosine, whichare complementary to the uracil, the conversion product of unmethylatedcytosine. Preferably, nucleotides that distinguish between the convertedmethylated and unmethylated sequences will be at or near the 3′ end ofthe primers. Variations of methods using sodium bisulfite-based PCR aredescribed in, e.g., Herman et al., PNAS USA 93:9821-9826 (1996); U.S.Pat. Nos. 5,786,146 and 6,200,756.

3. Hybrid Capture

In some embodiments, nucleic acid hybrid capture assays can be used todetect the presence and quantity of DNA cleaved by the restrictionenzymes. This method can be used with or without amplifying the DNA.Following the restriction digests, RNA probes which specificallyhybridize to DNA sequences of interest are combined with the DNA to formRNA:DNA hybrids. Antibodies that bind to RNA:DNA hybrids are then usedto detect the presence of the hybrids and therefore, the presence andamount of uncut DNA. DNA fragments that are restricted in a window ofsequence that is complimentary to the RNA probe hybridize lessefficiently to the RNA probe than do DNA fragments that remain in tactin the window of sequence being monitored. The amount of hybridizationallows one to quantify intact DNA, and the quantity of DNA methylationcan be inferred directly from the quantity of intact DNA from differentrestriction enzyme treatments (i.e., methylation-sensitive and/ormethylation-dependent restriction enzyme treatments).

Methods of detecting RNA:DNA hybrids using antibodies are known in theart and are described in, e.g., Van Der Pol et al., J. Clin. Microbiol.40(10): 3558 (2002); Federschneider et al., Am. J. Obstet. Gynecol.191(3):757 (2004); Pretet et al., J. Clin. Virol. 31(2):140-7 (2004);Giovannelli et al., J. Clin. Microbiol. 42(8):3861 (2004); Masumoto etal., Gynecol. Oncol. 94(2):509-14 (2004); Nonogaki et al., Acta Cytol.48(4):514 (2004); Negri et al., Am. J. Clin. Pathol. 122(1):90 (2004);Sarian et al., Gynecol. Oncol. 94(l):181 (2004); Oliveira et al., Diagn.Cytopathol. 31(1):19 (2004); Rowe et al., Diagn. Cytopathol. 30(6):426(2004); Clavel et al., Br. J. Cancer 90(9):1803-8 (2004); Schiller etal., Am. J. Clin. Pathol. 121(4):537 (2004); Arbyn et al., J. Natl.Cancer Inst. 96(4):280 (2004); Syrjanen et al., J. Clin. Microbiol.February 2004;42(2):505 (2004); Lin et al., J. Clin. Microbiol.42(1):366 (2004); Guyot et al., BMC Infect. Dis. 25;3(1):23 (2003); Kimet al., Gynecol. Oncol. 89(2):210-7 (2003); Negri et al., Am J SurgPathol. 27(2):187 (2003); Vince et al., J. Clin. Virol. Suppl 3:S109(2002); Poljak et al., J. Clin. Virol. Suppl 3:S89 (2002). In somecases, the antibodies are labeled with a detectable label (e.g., anenzymatic label, an isotope, or a fluorescent label) to facilitatedetection. Alternatively, the antibody:nucleic acid complex may befurther contacted with a secondary antibody labeled with a detectablelabel. For a review of suitable immunological and immunoassayprocedures, see, e.g., Harlow & Lane, ANTIBODIES, A LABORATORY MANUAL,Cold Spring Harbor Publication, New York (1988); Basic and ClinicalImmunology (Stites & Terr eds., 7^(th) ed. 1991); U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168); Methods in CellBiology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993).

Monoclonal, polyclonal antibodies, or mixtures thereof may be used tobind the RNA:DNA hybrids. Detection of RNA:DNA hybrids using monoclonalantibodies is described in, e.g., U.S. Pat. Nos. 4,732,847 and4,833,084. Detection of RNA:DNA hybrids using polyclonal antibodies isdescribed in, e.g., U.S. Pat. No. 6,686,151. The polyclonal ormonoclonal antibodies may be generated with specific binding properties.For example, monoclonal or polyclonal antibodies that specifically bindto shorter (e.g., less than 20 base pairs) or longer (e.g., more than100 base pairs) RNA:DNA hybrids may be generated. In addition,monoclonal or polyclonal antibodies may be produced that are either moreor less sensitive to mismatches within the RNA:DNA hybrid.

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with RNA:DNA hybrids are known to those of skill in theart. For example, preparation of polyclonal and monoclonal antibodies byimmunizing suitable laboratory animals (e.g., chickens, mice, rabbits,rats, goats, horses, and the like) with an appropriate immunogen (e.g.,an RNA:DNA hybrid). Such methods are described in, e.g., Coligan,Current Protocols in Immunology (1991); Harlow & Lane, supra; Goding,Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler& Milstein, Nature 256:495497 (1975).

Antibodies can also be recombinantly produced. Antibody preparation byselection of antibodies from libraries of nucleic acids encodingrecombinant antibodies packaged in phage or similar vectors is describedin, e.g., Huse et al., Science 246:1275-1281 (1989) and Ward et al.,Nature 341:544-546 (1989). In addition, antibodies can be producedrecombinantly using methods known in the art and described in, e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989);Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); andCurrent Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

D. Detection Of Methylation Density

In some embodiments, the methods of the invention can be used todetermine the methylation density of a locus. Determination ofmethylation density is described, e.g., in U.S. Patent Application No.60/513,426, filed Oct. 21, 2003.

The quantity of methylation of a locus of DNA can be determined byproviding a sample of genomic DNA comprising the locus, cleaving the DNAwith a restriction enzyme that is either methylation-sensitive ormethylation-dependent, and then quantifying the amount of intact DNA orquantifying the amount of cut DNA at the DNA locus of interest.

The amount of intact or cut DNA will depend on the initial amount ofgenomic DNA containing the locus, the amount of methylation in thelocus, and the number (i.e., the fraction) of nucleotides in the locusthat are methylated in the genomic DNA. The amount of methylation in aDNA locus can be determined by comparing the quantity of intact or cutDNA to a control value representing the quantity of intact or cut DNA ina similarly-treated DNA sample. As discussed below, the control valuecan represent a known or predicted number of methylated nucleotides.Alternatively, the control value can represent the quantity of intact orcut DNA from the same locus in another (e.g., normal, non-diseased) cellor a second locus.

By using at least one methylation-sensitive or methylation-dependentrestriction enzyme under conditions that allow for at least some copiesof potential restriction enzyme cleavage sites in the locus to remainuncleaved and subsequently quantifying the remaining intact copies andcomparing the quantity to a control, average methylation density of alocus may be determined. If the methylation-sensitive restriction enzymeis contacted to copies of a DNA locus under conditions that allow for atleast some copies of potential restriction enzyme cleavage sites in thelocus to remain uncleaved, then the remaining intact DNA will bedirectly proportional to the methylation density, and thus may becompared to a control to determine the relative methylation density ofthe locus in the sample. Similarly, if a methylation-dependentrestriction enzyme is contacted to copies of a DNA locus underconditions that allow for at least some copies of potential restrictionenzyme cleavage sites in the locus to remain uncleaved, then theremaining intact DNA will be inversely proportional to the methylationdensity, and thus may be compared to a control to determine the relativemethylation density of the locus in the sample.

The average methylation density within a locus in a DNA sample isdetermined by digesting the DNA with a methylation-sensitive ormethylation-dependent restriction enzyme and quantifying the relativeamount of remaining intact DNA compared to a DNA sample comprising aknown amount of methylated DNA. In these embodiments, partial digestionsor complete digestions can be used. As described above for uniformlymethylated DNA, use of partial digestions allows for the determinationof the average methylation density of the locus.

E. Detection of Methylation Differences Between Samples and at SpecificLoci

The methods of the invention can be used to detect differences inmethylation between nucleic acid samples (e.g., DNA or genomic DNA)and/or at specific loci. In some embodiments, the methods can be used toanalyze a sample of DNA where all copies of a genomic DNA locus have anidentical methylation pattern. In some embodiments, the DNA sample is amixture of DNA comprising alleles of a DNA locus in which some allelesare more methylated than others. In some embodiments, a DNA samplecontains DNA from two or more different cell types, wherein each celltype has a different methylation density at a particular locus (e.g., acell from a tissue suspected of being diseased and a cell from anon-diseased tissue sample). For example, at some loci, neoplastic cellshave different methylation densities compared to normal cells. If atissue, body fluid, or secretion contains DNA from both normal andneoplastic cells, the DNA sample from the tissue, body fluid, orsecretion will comprise a heterogeneous mixture of differentiallymethylated alleles. In this case, at a given locus, one set of alleleswithin the DNA (e.g., those derived from neoplastic cells in the sample)will have a different methylation density than the other set of alleles(e.g., those derived from normal cells).

In cases where a particular phenotype or disease is to be detected, DNAsamples should be prepared from a tissue of interest, or as appropriate,from blood. For example, DNA can be prepared from biopsy tissue todetect the methylation state of a particular locus associated withcancer. The nucleic acid-containing specimen used for detection ofmethylated loci (see, e.g., Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY (1995 supplement)) may be from any source includingbrain, colon, urogenital, hematopoietic, thymus, testis, ovarian,uterine, prostate, breast, colon, lung and renal tissue and may beextracted by a variety of techniques such as that described by Ausubelet al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1995) or Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (3rd ed. 2001).

Detection and identification of loci of altered methylation (compared tonormal cells) in DNA samples can indicate that at least some of thecells from which the sample was derived are diseased. Such diseasesinclude but are not limited to, e.g., low grade astrocytoma, anaplasticastrocytoma, glioblastoma, medulloblastoma, colon cancer, liver cancer,lung cancer, renal cancer, leukemia (e.g., acute lymphocytic leukemia,chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloidleukemia), lymphoma, breast cancer, prostate cancer, cervical cancer,endometrial cancer, neuroblastoma, cancer of the oral cavity (e.g.,tongue, mouth, pharynx), esophageal cancer, stomach cancer, cancer ofthe small intestine, rectal cancer, anal cancer, cancer of the analcanal and anorectum, cancer of the intrahepatic bile duct, gallbladdercancer, biliary cancer, pancreatic cancer, bone cancer, cancer of thejoints, skin cancer (e.g., melanoma, non-epithelial cancer, basal cellcarcinoma, squamous cell carcinoma), soft tissue cancers, uterinecancer, ovarian cancer, vulval cancer, vaginal cancer, urinary cancer,cancer of the ureter, cancer of the eye, head and neck cancer,non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, brain cancer,cancer of the nervous system. Identification of altered methylationprofiles is also useful for detection and diagnosis of loss of genomicimprinting, fragile X syndrome and X-chromosome inactivation.

Specific loci that are suitable for analysis using the methods of theinvention are described in, e.g., Costello and Plass, J. Med. Genet.38:285-303 (2001) and Jones and Baylin, Nature. Rev. 3:415-428 (2002)and are set forth in Table 1 below.

TABLE 1 Examples of Genes Exhibiting Hypermethylation in Cancer Effectof loss of function in tumor Gene development Tumor types RB Loss ofcell-cycle control Retinoblastoma MLH1 Increased mutation rate, drugColon, ovarian, endometrial, gastric resistance BRCA1 Genomicinstability Breast, ovarian E-CAD Increased cell motility Breast,gastric, lung, prostate, colon, leukemia APC Aberrant cell transductionBreast, lung, colon, gastric, esophageal, pancreatic, hepatocellular p16Loss of cell-cycle control Most tumor types VHL Altered proteindegradation Clear-cell renal cell carcinoma p73 Loss of cell-cyclecontrol Leukemia, lymphoma, ovarian RASSF1A Aberrant cell transductionLung, breast, ovarian, kidney, nasopharyngeal p15 Loss of cell-cyclecontrol Leukemia, lymphoma, gastric, squamous cell carcinoma,hepatocellular GSTP1 Increased DNA damage Prostate DAPK Reducedapoptosis Lymphoma, lung MGMT Increased mutation rate Colon, lung,brain, esophageal, gastric P14ARF Loss of cell cycle control Melanoma,non-melanoma skin cancer, pancreatic, breast, head and neck, lung,mesothelioma, neurofibromatosis, colon, soft tissue sarcoma., bladder,Hodgkin's, Ewing's sarcoma, Wilm's tumor, osteosarcoma, rhabdomyosarcomaATM Defective DNA repair Leukemia, lymphoma CDKN2B Loss of cell cyclecontrol Breast, ovarian, prostate FHIT Defective DNA repair Lung,pancreas, stomach, kidney, cervix, breast MSH2 Defective DNA repairColon NF1/2 Loss of cell cycle control Neurofibroma PTCH Loss of cellcycle control Skin, basal and squamous cell carcinomas, brain PTEN Lossof cell cycle control Breast, thyroid, skin, head and neck, endometrialSMAD4 Loss of cell cycle control Pancreas, colon SMARCA3/B1 Loss of cellcycle control Colon STK11 Loss of cell cycle control Melanoma,gastrointestinal TIMP3 Disruption of cellular matrix Uterus, breast,colon, brain, kidney TP53 Loss of cell cycle control; reduced Colon,prostate, breast, gall bladder, bile duct, apoptosis BCL2 Loss of cellcycle control; reduced Lymphoma, breast apoptosis OBCAM Loss of cellcycle control Ovarian GATA4 Transcriptional silencing of Colorectal,gastric, ovary downstream genes GATA5 Transcriptional silencing ofColorectal, gastric, ovary downstream genes HIC1 Loss of cell cyclecontrol Epithelium, lymphoma, sarcoma Abbreviations: APC, adenomatouspolyposis coli; BRCA1, breast cancer 1; DAPK, death-associated proteinkinase; E-cad, epithelial cadherin; GSTP1 glutathione S-transferase π1;MLH1, MutL homologue 1, MGMT, O(6)-methylguanine-DNA methyltransferase;p15, p15^(INK4b); p16, p16^(INK4); p73, p73; Rb, retinoblastoma;RASSF1a, Ras association domain family 1A; VHL, von Hippel-Lindau; ATM,ataxia telangiectasia mutated; CDKN2, cyclin dependent kinase inhibitor;FHIT, fragile histidine triad; MSH2, mutS homologue 2; NF½,neurofibromin ½; PTCH, patched homologue; PTEN, phosphatase and tensinhomologue; SMAD4, mothers against decapentaplegic homologue 4;SMARCA3/B1, SWI/SNF-related, matrix-associated, actin-dependentregulator of chromatin, subfamily A, member 3/subfamily B, member 1;STK11, serine/threonine kinase 11; TIMP3, tissue inhibitor ofmetalloproteinase 3; Bcl-2m B-call CLL/Lymphoma 2; OBCAM, opoid-bindingcell adhesion molecule; GATA, globin transcription factor; HIC1,hypermethylated in cancer.

In some embodiments, the methylation of sample from the same individualis determined over a period of time, e.g., days, weeks, months, oryears. Determination of changes in methylation can be useful forproviding diagnoses; prognoses; therapy selection; and monitoringprogression for various diseases; and, in the case of cancer, tumortyping and staging. While the methods of the invention also provide forthe detection of specific methylation events, the present methods areparticularly notable because they are not limited by a prediction orexpectation that the methylation state of a particular nucleotide isdeterminative of a phenotype. In cases where the density of methylation(rather than the presence or absence of a particular methylatednucleotide) modulates gene expression, and where the methylation densityof a locus reflects disease progression along a continuum, the presentmethods are particularly helpful.

Amplification primers can be designed to amplify loci associated with aparticular phenotype or disease.

If desired, multiplex DNA methods can be used to amplify multipletargets from the same sample. The additional targets can representcontrols (e.g., from a locus of known methylation status) or additionalloci associated with a phenotype or disease.

In some embodiments, the methods of the invention are used to identifynew loci associated with a disease phenotype, such as cancer, or areused to validate such an association.

F. Exemplary Methods of Determining Relative Methylation at a Locus

As described above, a number of possibilities are available fordetermining the relative amount of methylation at a genetic locus ofinterest. For example, partial or complete digestions can be performed,methylation-sensitive or methylation-dependent restriction enzymes canbe used, sodium bisulfite treatment can be employed, etc. Withoutintending to limit the invention to a particular series of steps, thefollowing possibilities are further exemplified.

In some embodiments, a DNA sample is digested (partially or tocompletion) with McrBC or another methyl-dependent restriction enzymeand a locus is subsequently amplified using quantitative DNAamplification (e.g., PCR, rolling circle amplification, and othermethods known to those skilled in the art). The resulting kineticprofiles of the amplification reactions are compared to those derivedfrom a similarly treated control DNA sample. Kinetic profiles ofamplification reactions can be obtained by numerous means known to thoseskilled in the art, which include fluorescence reaction monitoring ofTaqMan™, Molecular Beacons™, SYBRGREEN™ incorporation, Scorpion™ probes,Lux™ probes, and others.

In some embodiments, the DNA sample is split into equal portions and oneportion is treated with the methylation-dependent restriction enzyme andthe other is not. The two portions are amplified and compared todetermine the relative amount of methylation at the locus.

In some embodiments, the DNA sample can be split into equal portions,wherein each portion is submitted to a different amount of partialdigestion with McrBC or another methylation-dependent restrictionenzyme. The amount of intact locus in the various portions (e.g., asmeasured by quantitative DNA amplification) can be compared to a controlpopulation (either from the same sample representing uncut DNA orequivalent portions from another DNA sample). In cases where theequivalent portions are from a second DNA sample, the second sample canhave an expected or known number of methylated nucleotides (or at leastmethylated restriction enzyme recognition sequences) or, alternatively,the number of methylated recognition sequences can be unknown. In thelatter case, the control sample will often be from a sample ofbiological relevance, e.g., from a diseased or normal tissue, etc.

In some embodiments, the DNA sample is partially digested with one ormore methylation-sensitive restriction enzymes and then amplified toidentify intact loci. Controls in these cases are similar to those usedfor methylation-dependent restriction enzyme digestions described above.Untreated controls are undigested, and any treated control DNA samplesare digested with methylation-sensitive restriction enzymes.

In some embodiments, a sample is separated into at least two portions.The first portion is digested with an enzyme from one of the threepossible methylation-sensing classes (i.e., methylation sensitive,methylation insensitive, and methylation dependent). Each additionalportion is digested with the isoschizomeric partner from a differentmethylation-sensing class from the enzyme used to digest the firstportion. The intact loci are then amplified and quantified. The relativemethylation at the locus can be determined by comparing the resultsobtained from any two of the reactions to each other, with or withoutcomparison to an undigested portion. In the case where methylationinsensitive enzymes are used, the portion must undergo a partialdigestion.

In some embodiments, the DNA sample is treated with an agent thatmodifies unmethylated cytosine, but leaves methylated cytosineunmodified, e.g., sodium bisulfite. The sample is separated into equalportions, and one portion is treated with a methylation-dependentrestriction enzyme (e.g., McrBC). Sodium bisulfite treatment does notmodify McrBC recognition sites because sodium bisulfite modifiesunmethylated cytosine and the recognition site of each McrBC hemi-siteis a purine base followed by a methylated cytosine. Samples from bothcut and uncut portions are then amplified using at least one primer thatdistinguishes between methylated and unmethylated nucleotides. Theamplified portions are then compared to determine relative methylation.Certain quantitative amplification technologies employ one or moredetection probes that are distinct from the amplification primers. Thesedetection probes can also be designed to discriminate between convertedmethylated and unmethylated DNA. In some embodiments, the detectionprobes are used in combination with a methylation-dependent restrictionenzyme (e.g., McrBC). For example, the detection probes can be used toquantify methylation density within a locus by comparing the kineticamplification profiles between a converted McrBC treated sample and aconverted sample that was not treated with McrBC.

Alternatively, in some embodiments, the sample is divided into equalportions and one portion is digested (partially or completely) with amethylation-dependent restriction enzyme (e.g., McrBC). Both portionsare then treated with sodium bisulfite and analyzed by quantitativeamplification using a primer that distinguishes between convertedmethylated and unmethylated nucleotides. The amplification products arecompared to each other as well as a standard to determine the relativemethylation density.

In some embodiments, the DNA sample is divided into portions and oneportion is treated with one or more methylation-sensitive restrictionenzymes. The digested portion is then further subdivided and onesubdivision is digested with a methylation-dependent restriction enzyme(e.g., McrBC). The various portions and subportions are then amplifiedand compared. Following digestion, the portions and subportions canoptionally be treated with sodium bisulfite and amplified using at leastone primer that distinguishes between methylated and unmethylatednucleotides.

In some embodiments, the DNA sample is divided into four portions: afirst portion is left untreated, a second portion is contacted with amethylation-sensitive restriction enzyme (wherein intact sequences aremethylated), a third portion is contacted with a methylation-dependentrestriction enzyme (wherein intact sequences are unmethylated), and afourth portion is contacted with a methylation-sensitive restrictionenzyme and a methylation-dependent restriction enzyme in which one ofthe restriction enzymes in the fourth portion is contacted to the sampleunder conditions that allow for at least some copies of potentialrestriction enzyme cleavage sites in the locus to remain uncleaved(e.g., under partial digest conditions and/or using McrBC). If desired,a fifth portion of the sample can be analyzed following treatment with amethylation insensitive isoschizomer of a methylation-dependent ormethylation-sensitive restriction enzyme used in another portion,thereby controlling for incomplete digestions and/or mutations at therestriction enzyme recognition sequence. In addition to digestion, theportions and subportions can optionally be treated with sodium bisulfiteand amplified using at least one primer that distinguishes betweenconverted methylated and unmethylated nucleotides.

VIII. Kits

The present invention also provides kits for performing the methods ofthe invention. For example, the kits of the invention can comprise,e.g., a methylation-dependent restriction enzyme or a methylationsensitive restriction enzyme, a control DNA molecule comprising apre-determined number of methylated nucleotides, and one or twodifferent control oligonucleotide primers that hybridize to the controlDNA molecule. In some cases, the kits comprise a plurality of DNAmolecules comprising different pre-determined numbers of methylatednucleotides to enable the user to compare amplification of a sample toseveral DNAs comprising a known number of methylated nucleotides.

The kits of the invention will often contain written instructions forusing the kits. The kits can also comprise reagents sufficient tosupport the activity of the restriction enzyme. The kits can alsoinclude a thermostable DNA polymerase.

In some cases, the kits comprise one or two different targetoligonucleotide primers that hybridize to a pre-determined region ofhuman genomic DNA. For example, as described above, the primers canallow for amplification of loci associated with the development orprognosis of disease.

In some embodiments, the kits may comprise one or moredetectably-labeled oligonucleotide probes to monitor amplification oftarget polynucleotides.

In some embodiments, the kits comprise at least one targetoligonucleotide primer that distinguishes between modified unmethylatedand methylated DNA in human genomic DNA. In these embodiments, the kitsalso typically include a fluorescent moiety that allows the kineticprofile of any amplification reaction to be acquired in real time.

In some embodiments, the kits comprise at least one targetoligonucleotide primer that distinguishes between modified unmethylatedand methylated DNA in human genomic DNA. In these embodiments, the kitswill also typically include an agent that modifies unmethylatedcytosine.

In some embodiments, the kits comprise an RNA probe, a binding agent(e.g., an antibody or an antibody mimetic) that specifically bindsRNA:DNA complexes, detection reagents, and methylation sensitive and/ormethylation dependent restriction enzymes.

EXAMPLES Example 1 Comparison to Identify Methylation at a Locus

The following example illustrates how the methods of the invention canbe used to determine the number of methylated copies of a locus, thenumber of unmethylated copies of a locus, the number of hemimethylatedcopies of a locus, the number of mutated copies of a locus, and thetotal number of copies of a locus in a population of nucleic acids. Thenucleic acids are isolated from a sample and divided into portions, eachof which contain all of the sequences present in the sample. Thisexample uses the restriction site for Sau3A I for illustrative purposesand monitors 6 mA methylation. One of skill in the art will appreciatethat different enzymes could be selected to monitor cytosinemethylation.

Any given restriction site has three potential states: (1)hemimethylated; (2) methylated; (3) unmethylated; and (4) mutated.

1. G*ATC = hemimethylated (“hemi”) CTAG 2. G*ATC = methylated (“meth”)CTA*G 3. GATC = unmethylated (“unmeth”) CTAG 4. GTTC = mutated (“mut”)CAAG

Sau3A I is a methylation insensitive restriction enzyme which cuts whena methylated adenosine residue is at its recognition site. Dpn I is amethylation-dependent restriction enzyme which cuts only when amethylated adenosine residue is at or near its recognition site on bothstrands. Mbo I is a methylation-sensitive enzyme that does not cut whena methylated adenosine residue is at its recognition site, and is alsothe isoschizomer of Sau3A I. Hemimethylated sites are cut by Sau3A I,but not by Dpn I or Mbo I; methylated sites are cut by Dpn I and Sau3AI, but not by Mbo I; and unmethylated sites are cut by Sau3A I and MboI, but not by Dpn I.

A. Quantitative PCR of a first portion of untreated nucleic acids, ormock treated nucleic acids, from the sample yields the total number ofcopies of the locus in the sample, which equals:(1) hemi+(2) meth+(3) unmeth+(4) mut.B: Cutting a second portion of nucleic acids with themethylation-sensitive restriction enzyme Mbo I, followed by quantitativePCR, leads to amplification of the methylated copies of the locus in thesample, which equals:(1) hemi+(2) meth+(4) mut.C: Cutting a third portion of nucleic acids cut with themethylation-dependent restriction enzyme Dpn I followed by quantitativePCR, leads to amplification of the unmethylated copies in the sample,which equals:(1) hemi+(3) unmeth+(4) mut.D. Cutting a fourth portion of nucleic acids with themethylation-sensitive restriction enzyme Mbo I, and themethylation-dependent restriction enzyme Dpn I, followed by quantitativePCR leads to leads to amplification of hemimethylated copies in thesample, which equals:(1) hemi+(4) mut.E: Cutting a fifth portion of nucleic acids cut with Sau3A I, amethylation-insensitive restriction enzyme that is an isoschizomer ofthe methylation-dependent restriction enzyme (Dpn I), followed byquantitative PCR, leads to amplification of mutant copies in the sample,i.e., copies which are complementary to the PCR primers, but do notcontain hemimethylated, methylated, or unmethylated restriction sites,which equals:(4) mut.F. A comparison of the results from A and B leads to the number ofunmethylated loci in the sample:Unmeth=A[hemi+meth+unmeth+mut]−B[hemi+meth+mut].G. A comparison of the results from A and C leads to the number ofmethylated copies in the sample:Meth=A[hemi+meth+unmeth+mut]−C[hemi+unmeth+mut].H. A comparison of the results from A, B, and C leads to the number ofhemimethylated copies and unmethylated copies in the sample:Hemi+unmeth=C[hemi+unmeth+mut]−(A[hemi+meth+unmeth+mut]−B[hemi+meth+mut]).Hemi+unmeth=B[hemi+meth+mut]−(A[hemi+meth+unmeth+mut]−B[hemi+unmeth+mut]).I. A comparison of the results from A and D leads to the number ofmethylated and unmethylated copies in the sample:Meth+unmeth=A[hemi+meth+unmeth+mut]−D[hemi+mut].J. A comparison of the results from D and E leads to the number ofhemimethylated copies in the sample:Hemi=D[hemi+mut]−E[mut].K. A comparison of the results from E and D with B or C leads to thenumber of methylated or unmethylated copies in the sample, respectively:Meth=B[hemi+meth+mut]−E[mut]−(D[hemi+mut]−E[mut])Unmeth=C[hemi+unmeth+mut]−E[mut]−((D[hemi+mut]−E[mut]).

Example 2 Demonstrating the Sensitivity of Detection

Human male placental DNA was obtained and was methylated in vitro usingM.SssI, which methylates cytosines (5mC) when the cytosines are followedby guanosine (i.e., GC motifs). The resulting in vitro methylated DNAwas then mixed into unmethylated male placental DNA at known ratios,thereby producing a set of mixes, each comprising a different percentageof total copies that are methylated.

The various mixtures were then divided into three portions: an uncutportion; a portion digested with HhaI, a methylation-sensitiverestriction enzyme that is sensitive to 5mC and having the recognitionsequence GCGC, where underlined nucleotides are unmethylated; and aportion digested with both HhaI and McrBC. McrBC is amethylation-dependent restriction enzyme that cleaves in the proximityof its methylated recognition sequence. The digested sequences weresubsequently amplified using primers specific for a region upstream ofthe CDKN2A (p16) gene in the human genome-[Ensembl gene ID#ENSG00000147889]. This region was determined to be unmethylated in humanmale placental DNA that has not been methylated in vitro. The primersequences were:

Forward primer 5′-CGGGGACGCGAGCAGCACCAGAAT-3′ (SEQ ID NO: 2), Reverseprimer 5′ CGCCCCCACCCCCACCACCAT-3′ (SEQ ID NO: 3)and standard PCR conditions were used:

1 cycle [at 95° C. for 3 minutes]

followed by 49 cycles at [95° C. for 30 sec, 65° C. for 15 seconds, and68° C. for 15 seconds, a plate read (68° C.) and then another plate readat 83° C.]. The second plate reading at 83° C. was conducted toeliminate the fluorescence contribution of primer dimers to the reactionprofile. A melt-curve, which measures fluorescence as a function oftemperature, was performed between 80° C. and 95° C. at the end of thecycles and product specificity was determined. The locus of interest is181 bp in length and has a melting temperature of approximately 89° C.Amplification product accumulation was determined using theintercalating dye, SYBR Green™ Dynamo Kit from MJ Research, whichfluoresces when it binds to double stranded nucleic acids, and reactionswere cycled and fluorescent intensity was monitored using the MJ OpticonII Real-time PCR machine.

A threshold at which the signal from the amplification products could bedetected above background was determined empirically from a parallelanalysis of a template dilution standard curve. The threshold wasadjusted to maximize the fit of the regression curve (Ct vs log [DNA]),according to standard cycle threshold determination protocols familiarto those skilled in the art, such as those described in e.g., Fortin etal., Anal. Biochem. 289: 281-288 (2001). Once set, the threshold wasfixed and the Ct was calculated by the software (MJ Research Opticon IIMonitor V2.02). As expected, the derived cycle thresholds increased athigher dilutions of methylated to unmethylated DNA (FIG. 2). Also shownin FIG. 2, the change (or “shift”) in cycle threshold (ΔCt) betweenuncut DNA and the HhaI treated DNA corresponded with that expected (E)for the dilutions, demonstrating that cycle threshold shift can be usedto accurately predict the relative proportion of copies that aremethylated in the sample out of the total number of copies in thesample.

FIG. 2 also illustrates that the addition of HhaI (a methylationsensitive restriction enzyme) and McrBC (a methylation-dependentrestriction enzyme) further alters the Ct compared to the samplestreated with HhaI alone. The degradation in the number of intact copies,and the resultant Ct shift to a higher Ct value after treatment with themethylation dependent restriction enzyme and the methylation sensitiveenzyme further confirms the assessment that the intact copies presentafter treatment with the methylation-sensitive restriction enzyme aloneare in fact methylated. In other words, this double digest provides acontrol against the possibility that HhaI was not added, was inactive,was partially active, or otherwise did not result in a complete digest.The addition of the methylation-dependent restriction enzyme and itsability to destroy methylated templates confirms the results observedafter treatment with just the methylation-sensitive restriction enzymeand provides an internal control to assess the completeness of themethylation-sensitive restriction enzyme reaction.

FIG. 3 depicts the kinetic profile of four portions at three dilution ofmethylated DNA to unmethylated DNA. In each of the three dilutions, allfour portions were digested first with the methylation sensitiverestriction enzyme HhaI. The first two portions in each dilution weredigested with McrBC, and the second two portions in each dilution wereuntreated with regards to McrBC. All portions were the amplified underidentical conditions and the fluorescence intensities were measured.Three observations can be made. First, the duplicate reactions havenearly identical Ct values, demonstrating that the assays are highlyreproducible. Second, decreasing change in cycle threshold betweentreated and untreated portions as a function of increasing dilution ofthe methylated copies shows that as the methylated gene copies get morerare, there is less difference between the Ct values observed betweenMcrBC treated and untreated portion. This suggests that the Hha andHha+McrBC reactions will converge and that at some point we will not beable to monitor methylation density or be able to identify the presenceor absence of methylated copies. A theoretical extinction of detectionwill occur at a ΔCt of zero. Using a regression analysis we solved forthe extinction function in our system and found that the dilution whereΔCt=0 is 1:20,000, methylated copies to unmethylated copiesrespectively. This regression analysis is detailed in FIG. 5.

FIG. 3 shows the fluorescent kinetic profile of a series of portions alldiluted to 1:2,000 methylated copies to unmethylated copies,respectively. While the overall fluorescence obtained from the 1:2,000reactions is not ideal, one can see a difference between the HhaI andthe HhaI/McrBC reactions. Notice that the McrBC digestion destroys theaccumulation of the fluorescence, and the melting point curve in FIG. 4shows a specific peak at 89° C., which is the predicted meltingtemperature for the 181 bp specific amplicon. Here we are clearlydetecting merely 1.4 cellular equivalents of methylated DNA diluted intoa total of 2,762 cellular equivalents of DNA.

As shown in FIG. 5, 1.4 cellular equivalents (CE) were detected out of atotal of 2764 CE in the tube having a total of 20 ng of genomic DNA.Each cellular equivalent has approximately 7.9 pg of genomic DNA/percell. Thus, if 50 ng of genomic DNA is used, one methylated copy in thepresence of 10,000 unmethylated copies should be detectable. Thisprinciple is illustrated in FIG. 5. FIG. 6 provides a breakdown of thisanalysis. Note that this detection limit may be further lowered by (i)using an optimized FRET-based probe, rather than an intercalating dye,to detect amplified products, (ii) by further optimizing PCR primerdesign, or (iii) by further optimizing PCR reaction conditions.

Example 3 Constructing a DNA Methylation Standard Sample Set

A standard sample set is generated in numerous ways. For example, amethylase (e.g., M.SssI or other methylases such as M.HhaI, M.AluI) isapplied in vitro to a series of DNA samples to produce a standard set ofDNAs known to have increasing methylation densities. This standard setis generated by first obtaining a sample of known sequence (e.g., thelocus of interest). Next, the sample is divided into a series of samplesand each sample in the series is treated with the chosen methylase inthe presence of magnesium and in a manner that results in increasingmethylation densities of the samples in the series.

A partial methylation reaction refers to contacting DNA with a cocktailof one or more methylases under appropriate reaction conditions suchthat the methylase modifies some (e.g., about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%) but not all of the possible methylase recognitionsites for each enzyme in the methylase cocktail. A DNA sequence ispartially methylated by treating DNA with an active methylase for ashorter period of time than is necessary to achieve completemethylation, and then terminating the reaction, or under other alteredreaction conditions that allow for the desired amount of partialmethylation.

The methylation densities of each sample in the series are measured bysequencing a statistically significant sample of clones from abisulfite-treated portion of each series member in the set, byidentifying the converted cytosines within each clone, and bycalculating the average methylation density for each reaction within themethylation sample set. In order to achieve a partial methylationdensity on a given fragment, the methylase acts in a stochastic manner,and not a processive manner. For M.SssI, this is achieved by conductingthe reaction in the presence of magnesium, since M.SssI methylates DNAin a processive way in the absence of magnesium, while in the presenceof magnesium, the enzyme methylates CpGs in a nonprocessive, stochasticmanner.

Example 4 Quantitatively Determining the Relative Methylation of a Locusof Interest between One Tissue and Another Tissue with QuantitativeAmplification

DNA is collected from two sources: a test population (diseased) and acontrol population (normal).

Each population of DNA fragments is similarly submitted to variouspartial or complete digestions with the enzyme McrBC. McrBC recognizestwo R^(M)C sites, each a half site, that are within 20 to 4,000 baseswith an optimal separation of the half sites of 50-103 bp and then cutsthe DNA fragment sometimes 3′ of both half sites, sometimes 3′ of the 5′most half site and 5′ of the 3′ most half sites, and sometimes 5′ ofboth half sites.

Next, the digested DNA in each population is amplified and the amount ofthe amplified locus is measured as a function of cycle number. Thegreater the number of methylated half sites in the locus of interest ona given DNA fragment within the population studied, the greater theprobability that McrBC will cut between the PCR primers, and, therefore,a greater number of amplification cycles will be required to achieve theidentical concentration of amplified PCR locus.

To determine whether the locus of interest within the test population ismore or less methylated than the locus of interest within the controlpopulation, a concentration curve of amplified DNA of the testpopulation is compared to the concentration curve of amplified DNA fromthe control population. Concentration curves reflect the amount ofintact DNA as a function of the amount of digestion in a series ofdifferent partial digestions.

Example 5 Measuring the Methylation Density at a Locus of Interestwithin a Tissue with Quantitative Amplification

DNA is obtained from a single source, and is divided into twopopulations. The first population of DNA is completely digested with theenzyme McrBC, while the remaining population is untreated.Alternatively, the first population is digested with a cocktail of oneor more methylation sensitive restriction enzymes (e.g., HpaII, HhaI, orAciI, etc.), while the second population of DNA is untreated.

Next, the digested DNA in the first population is amplified and theamount of the amplified locus is measured as a function of cycle number.The greater the number of methylated half sites in the locus of intereston a given DNA fragment within the population studied, the greater theprobability that McrBC will cut between the PCR primers, and, therefore,a greater number of amplification cycles will be required to achieve theidentical concentration of amplified PCR locus. Alternatively, when acocktail of methylation sensitive restriction enzymes is used, thegreater the number of methylated restriction sites in the locus ofinterest on a given DNA fragment within the population studied, thelower the probability that the methylation sensitive cocktail of enzymeswill cut between the PCR primers. Therefore, a lower number ofamplification cycles will be required to achieve the identicalconcentration of amplified PCR locus.

To determine whether the locus of interest within the first populationis methylated, a comparison is made between the kinetics of theamplification reaction profiles from the treated and untreatedpopulations. Alternatively, to determine the density of methylationwithin the tissue at the locus of interest, the kinetics of theamplification reaction profiles are compared to those obtained from aknown in vitro generated methylation sample set, i.e., a standardmethylation curve.

Example 6 Measuring the Methylation Density at a Locus of Interestwithin a Tissue with Amplification End Point Analysis

DNA is obtained from a single source, and is divided into a series oftwo or more portions.

This series is exposed to an increasing amount of partial digestion by amethylation dependent restriction enzyme, such as McrBC. The firstportion of DNA fragments is untreated, the second portion is lightlydigested with McrBC, and subsequent populations are more fully digested(but less than completely) with McrBC. The range of partial digestionsis obtained through the manipulation of reaction conditions, such as thetitration of enzyme amounts, digestion times, temperatures, reactants,buffers, or other required components.

Next, the DNA from the series of portions are amplified and the amountof amplified PCR loci is measured after a fixed number of cycles. Thegreater the number of methylated half sites in the locus of interest ona given DNA fragment within the first McrBC-treated portion, the greaterthe probability that McrBC will cut fragments of the first part betweenthe PCR primers, and the greater number of amplification cycles will berequired to detect a certain concentration of amplified PCR locus in thefirst portion.

To determine whether the locus of interest within the test population ismore or less methylated, the results obtained from the series ofportions and the parallel analysis of the standard sample set arecompared (see Example 3).

Example 7 Quantifying Methylation Using Methylation-SensingIsoschizomeric Partners and Quantitative PCR

DNA is collected from two sources: a test population (diseased) and acontrol population (normal). Each population is divided into groups oftwo or more portions. Each group is exposed to an increasing amount ofpartial digestion by a methylation sensitive restriction enzyme (e.g.,HpaII, MboI (A)). The first portion of DNA fragments is untreated, thesecond portion is lightly digested with the methylation sensitiverestriction enzyme, and subsequent populations are more fully digested(but less than to completion) with the enzyme. The range of partialdigestions is obtained through the manipulation of reaction conditions,such as the titration of enzyme amounts, digestion times, temperatures,reactants, buffers, or other required components.

The second group of portions is similarly digested with anisoschizomeric partner of a different methylation-sensing class from theenzyme used to treat the first group of portions (e.g., MspI and Sau3AI(A), respectively). Alternatively, the second group of portions remainsuntreated.

Next, all of the portions in the groups are amplified and the kineticreaction profile from each amplification is obtained. Alternatively, endpoint analysis after a fixed number of cycles is used.

To determine whether the locus of interest within the test population ismore or less methylated, a comparison is made between the kineticreaction profiles between the groups (group vs. group). Additionally, todetermine whether the locus of interest between the two tissues is moreor less methylated, a comparison is made between the kinetic reactionprofiles between the populations (diseased groups vs. normal groups).

Example 8 Quantifying the Methylation Density of a Locus of InterestUsing a Cocktail of Methylation Sensitive Enzymes

DNA is obtained from a single source and is divided into groups of twoor more portions. Alternatively, DNA is collected from two sources: atest population (diseased) and a control population (normal), and isdivided into groups of two or more uniform portions.

The groups of uniform portions are treated with a fixed number of unitsof a cocktail of one of more methylation sensitive restriction enzymes(e.g., HpaII, HhaI, or AciI) for varied amounts of time.

Next, all of the portions in the groups are amplified and the kineticreaction profile from each amplification is obtained. Alternatively, endpoint analysis after a fixed number of cycles is used.

To determine whether the locus of interest within the test population ismore or less methylated, a comparison is made between the kineticreaction profiles between the groups (group vs. group). Additionally, todetermine whether the locus of interest between the two tissues is moreor less methylated, a comparison is made between the kinetic reactionprofiles between the populations (diseased groups vs. normal groups).Finally, the overall amount of methylation can be determined bycomparing these results to those obtained from the standard sample set(see Example 3).

Example 9 Quantifying the Methylation Density of a Small Population ofMethylated Alleles in the Presence of a Large Population of UnmethylatedAlleles

DNA is obtained from a single source and is divided into two portions.Alternatively, DNA is collected from two sources: a test population(diseased) and a control population (normal), and each population isdivided into two portions.

To discriminate between methylated and unmethylated alleles, one portionfrom each population is treated with sodium bisulfite, which convertsthe unmethylated cytosine residues to uracil, leaving unconvertedmethylated cytosine residues. The bisulfite-treated portion is dividedinto two equal subportions. Alternatively, one portion from eachpopulation is digested with a cocktail of one or more methylationsensitive restriction enzymes (e.g., HpaII, HhaI, etc.), leaving theremaining portion untreated. The digested portion is similarly dividedinto two equal subportions.

One of the bisulfite-treated subportions is completely digested with theenzyme McrBC, while the remaining subportion is untreated.Alternatively, one of the methylation sensitive restrictionenzyme-treated subportions is completely digested with the enzyme McrBC,while the remaining subportion is untreated.

One or both of the amplification primers are designed to resemble thebisulfite converted sequence overlapping at least one methylatedcytosine residue. In this way, only those fragments that belong to thesubset of fragments that were methylated at that primer in the testpopulation have the potential of becoming amplified, while thosefragments in the subset of fragments that remained unmethylated in thelocus of interest will not be amplified. Alternatively, if methylationsensitive enzymes are used to discriminate between methylated andunmethylated alleles, then primers designed to the native sequence areused and only alleles that were methylated at the recognition sitesremain intact and will be amplified.

Next, the DNA from both the McrBC-treated and McrBC-untreated portions,along with the relevant controls, are amplified and the amount ofamplified PCR loci are measured as a function of cycle number.

To determine whether the locus of interest within the first populationis methylated, a comparison is made between the kinetics of theamplification reaction profiles from the treated and untreatedpopulations. To determine the density of methylation within the tissueat the locus of interest, the kinetics of the amplification reactionprofiles are compared to those obtained from a known in vitro generatedmethylation sample set, i.e., a standard methylation curve.

Alternatively, this Example could also be performed by reversing theorder of the sodium bisulfite conversion and the McrBC-digestion stepsdescribed above (i.e., McrBC digestion takes place prior to sodiumbisulfite conversion).

In another alternative, partial digestion using McrBC is used in eithera subportion or a series of subportions, instead of complete digestion.

Example 10 Detecting Methylation Density

This example demonstrates determining the average density of methylation(i.e., the average number of methylated nucleotides) within a locus. Asprovided in FIG. 7, it is likely that in many diseases, methylation ofone or more loci goes through a progression of increased methylationdensity corresponding to disease progression. Previously-describedmethylation detection techniques involve detecting the presence orabsence of methylation at one or more particular nucleotides but do notprovide analysis of density across a locus. In contrast, the presentinvention provides methods for detecting the average number ofmethylation events within a locus.

As illustrated in FIGS. 12-13, methods that detect methylation at onlyspecific short sequences (typically relying on primer or probehybridization) may miss changes in methylation (see FIG. 12) that thepresent methylation density detection methods (which examine relativemethylation across an entire locus) are able to detect (see FIG. 13).

This discovery works by treating a locus with a methylation-dependent ormethylation-sensitive restriction enzyme under condition such that atleast some copies of potential restriction enzyme cleavage sites in thelocus to remain uncleaved. While these conditions can be achieved byallowing for partial digestion of a sample, the particular recognitionand cutting activity of McrBC allows for additional options.

As discussed above, when two McrBC complexes meet, restriction occurs,typically within ˜32 bp of either half site (i.e., in one of fourregions). See, FIG. 8. Restriction does not occur if half sites arecloser than 20 bp.

Since McrBC randomly cuts 5′ or 3′ of the recognized pair of half sites,the probability of cutting at a locus (spanned by primers in the casePCR) is function of the number of methylated half-sites present at ornear the locus. For a set concentration of enzyme and time ofincubation, the more methylation sites within a locus, the greater theprobability McrBC will cut at the locus (or between the primers in thecase of PCR). However, under ideal circumstances and sufficient numberof DNA copies, the probability that McrBC will cut every copy of a locusis low because it will sometimes cut at a distance outside of the locus,thereby leaving the locus intact. Thus, the number of intact loci isinversely proportional to the average number of methylated nucleotideswithin the locus. The number of intact loci is inversely proportional tothe Ct value for sample. Thus, the Ct value is proportional to theaverage number of methylated nucleotides within a locus. Thus,comparison of the Ct value of amplified McrBC-treated DNA compared tothe Ct value from amplified untreated DNA allows for the determinationof methylation density of the locus.

Two aliquots of BAC DNA containing the p16 locus was in vitro methylatedat different densities. The first aliquot was densely methylated withM.sssI. There are 20 M.sssI methylase sites within the PCR amplicon, 11of which are also McrBC half-sites. The second aliquot was sparselymethylated with M.HhaI. There are four M.HhaI methylase sites within thePCR amplicon, all four of which are also McrBC half-sites. Within thePCR amplicon there are also 4 restriction sites for HhaI. All four ofthese HhaI restriction sites are methylase sites for both M.sssI andM.HhaI, such that complete treatment with either methylase will protectall four HhaI sites from restriction. A different number of units ofMcrBC was used for a set period of time (four hours) to generate aseries of progressively more partial digestions to identify an amount ofenzyme to best allow for distinguishing results from the sparsely anddensely-methylated DNA. As displayed in FIGS. 9 and 10, the Ct valueswere proportional to the concentration of McrBC used in both sparselyand densely-methylated sequences. FIG. 11 demonstrates results fromtitrating different amounts of McrBC to enhance resolution betweensparsely and densely methylated sequences to distinguish between thetwo. In FIG. 11, “1×” equals 0.8 units of McrBC.

The densely methylated target has 2.75-fold more methylated McrBC halfsites than the sparsely methylated target (11/4=2.75). Therefore, upontreatment with McrBC and subsequent amplification, we expect to see adifferent between the Ct of the reactions of about 1.46, because2^(ΔCt)=2.75. We observed ΔCt (sparse-dense @ 1×McrBC) was 1.51±0.05.Thus, the methylation density of a locus was determined using thismethod.

Example 11 Methylation Density Determination

This example demonstrates the ability of methylation-dependent andmethylation-sensitive restriction enzymes to distinguish differentmethylation densities at a locus.

A 703 bp portion of the promoter of p16 was amplified. The portion wasmethylated in vitro in a time course with M.SssI under conditions thatpromote stochastic methylation. The portion is illustrated in FIG. 14.From the large methylation reaction at different time points, fixedvolumes (20 μl) were removed and the methylation reactions were stoppedwith heat (65° C.). We stopped one reaction before it began (T=1 is 0minutes methylation, i.e., the unmethylated control; T=2 was stopped at2 minutes; T=3 was stopped at 5 minutes; and T=4 was stopped at 60minutes, a time at which the PCR product in the reaction should havebeen fully methylated.

The reactions were purified and each amplicon then was diluted more than1 million fold in TE buffer, and was added back to the human genome at aratio that should approximate a normal copy balance (i.e., two copiesper 7.9 picograms). The human genome used was homozygous for a deletionof the p16 gene. The deletion cell line is CRL-2610. This allowed us toadd a fixed amount of the human genome (i.e., control for the complexityof the genome in our reaction).

DNA samples were cleaved with Aci I (a methylation-sensitive restrictionenzyme), McrBC (a methylation-dependent restriction enzyme), or both asdouble digest, and the portion was amplified. Amplicons were detectedwith the MS_p16(207) SYBR green real-time PCR system. Twenty nanogramsof input DNA (genome +amplicon) equal ˜2764 cellular equivalents/per PCRreaction. Each set of four digests was brought up to volume inrestriction salts with BSA and GTP such that it could be split into fourtubes (˜4 μg). Each of the four digest tubes (˜1 μg) had 100 μl totalvolume such that 2 μl could be added to PCR reactions, thereby adding 20ng of DNA. Digests were allowed to proceed for four hours and were heatkilled for 20 minutes. PCR conditions:

The forward primer was CAGGGCGTCGCCAGGAGGAGGTCTGTGATT (SEQ ID NO: 4).The reverse primer was GGCGCTGCCCAACGCACCGAATAGTTACGG (SEQ ID NO: 5).

Dynamo MJ qPCR buffer, 65° C. anneal, two cycle PCR (95° C. 30 sec, 65°C. 20 sec) cycled 49 times and monitored with an MJ opticon IIquantitative PCR system.

We hypothesized that if the technology is monitoring density:

-   a) The McrBC cleavage should demonstrate a larger ΔCt for each    sample in a progression from 0 ΔCt for T=1, up to a maximal ΔCt at    T=4 (60 minutes)-   b) The Aci I reactions should demonstrate the converse relationship.-   c) The mock treated and double digests should be fixed reference    points

As illustrated in FIG. 15, we observed the trends outlined above. TheMcrBC curve moves oppositely from the Aci I curve, and the movement isproportional with the increasing methylation content in the locus. Theuntreated and double digests indicate the boundaries of the assay field.The system resolves the difference between each of the reactions alongthe time course, such that each graphical depiction showing the varioustimed reactions is different. The point where the profile intersects thedashed threshold line indicates the point where information is compared.

Another way to visualize the data is by plotting the change in cyclethreshold values (ΔCt). See, FIG. 16. FIG. 16 displays the ΔCt forMcrBC-treated compared to untreated at each time point in the partialmethylation reaction, and the corresponding ΔCt for the Aci I digests(Aci I compared to untreated). As expected, the McrBC and Aci I ΔCtlines provide an intersecting inverse pattern. The Aci I graph displaysa chunky shape because its cut sites are fixed, while McrBC displays asmooth continuous distribution, reflecting its ability to cut more orless randomly following site recognition. Frequency of cutting isproportional to the expected change in methylation occupancy based uponthe time course. The error bars associated with the real-timemeasurements are indicated. If they are not visualized, they are withinthe data point.

Example 12 Monitoring DNA Methylation of a Target Sequence Present atMultiple Locations in the Genome

This example demonstrates the ability of the present technology todetermine the methylation of a target sequence that is present in agenome more than one time (i.e., more than one copy) using an assay thatmonitors a sequence repeated in the kafirin gene cluster in Sorgumbicolor.

Eleven kafirin genes were annotated from the publicly available sequenceof a BAC clone AF527808 from Sorghum bicolor. PCR primers were designedto amplify a 247 bp amplicon from all 11 kafirin genes (the primersequences were conserved in all 11).

The forward primer was 5′ CTCCTTGCGCTCCTTGCTCTTTC 3′ (SEQ ID NO: 6). Thereverse primer was 5′ GCTGCGCTGCGATGGTCTGT 3′ (SEQ ID NO: 7).

Sorghum genomic DNA isolated from seedling leaf was divided into 6 equalportions. The six portions were treated in the following manner: i)untreated (mock treated), ii) HhaI digested, iii) McrBC digested, iv)HhaI and McrBC digested, v) PstI digested and, vi) PstI and McrBCdigested. Equal volume aliquots from the six portions were amplifiedusing the above PCR primers in the following manner:

The SYBR green real-time PCR cycling parameters were 95° C. for 3minutes, followed by 50 cycles of 2 step PCR 95° C. for 30 sec, 56° C.for 30 seconds with the Dynamo Kit from MJ Research (Boston, Mass.). Weutilized both a low temperature (70° C.) and a high-temperature plateread (82° C.). The input of genomic DNA was 10 ng per PCR reaction. Thethreshold was set using a template dilution standard control.

The kinetic profiles for the 6 PCR reactions are depicted in FIG. 18.The inset in FIG. 18 depicts the template dilution standard curve usedto set the cycle threshold for the experiment. Each set of 6 digests wasperformed three times, and each of the 18 digests had four PCRreplicates. The PCR reactions were determined to be highly reproducible.In FIG. 18, PCR amplification reaction kinetics for each of the sixdigestions are depicted with different colors: Red =mock treated,Blue=McrBC digested, Orange=HhaI digested, and Green=HhaI+McrBC doubledigest, Pink=PstI, and Azure=PstI+McrBC double-digest. Comparisonsbetween the cycle thresholds of the six amplified digestions were madeand of the density of CNG and CG methylation in the repeated targetsequence was determined.

In all 11 kafirin genes, all PstI sites in the repeated target sequencewere methylated (at CNG) and PstI digestion was blocked since the PstItreated sample (pink) has the same cycle threshold (Ct) as the mocktreated sample (red). This result is supported by the McrBC digestedsample (blue), which has a significantly higher Ct than themock-digested DNA control (red), further demonstrating that CNGmethylation exists because McrBC was able to cut, thereby lowering thenumber of intact copies of the target sequence. All, or almost all, ofthe PstI sites are methylated because the double PstI+McrBC digest(light blue) has the same Ct as McrBC alone (blue). Note that the McrBCdigestion with and without PstI yields the same Ct, while HhaI withMcrBC (green) yields a higher Ct on average; suggesting that not allHhaI sites were methylated and that HhaI was able to reduce the numberof intact copies of the target sequence. These results indicate thatevery target sequence has high CNG methylation covering all PstI sites,while some but not all HhaI sites are methylated, indicating partial CGmethylation of HhaI sites in the target sequence. The specificity ofeach reaction was confirmed using melt-curve analysis.

For the kafirin genes, the average difference in Ct between the McrBCsingle and HhaI+McrBC double digests is 2.46 cycles (22.08±0.34HhaI+McrBC−19.62±0.19 McrBC). We compared the cycle-thresholds ofgenomic DNA that had been subjected to various treatments and inferredmethylation occupancy through the changes in Ct mediated by thetreatments. The Ct of any locus is a function of the number of copiespresent within the assay tube. Each of the eleven genes was broken into˜1.5 kb pieces which were aligned to create a consensus kafirin assembly(FIG. 17). The consensus kafirin sequence was examined and PCR primersamplifying a 247 bp amplicon were selected (see above).

As for CG methylation, the HhaI digested (orange) sample has the same Ctas the mock treated control (red); however, the HhaI+McrBC double digest(green) has a Ct that is 2.46 cycles greater than the McrBC alone(blue), indicating that some HhaI sites must not be modified. A cyclethreshold difference of 2.46 indicates that there is 2^(2.46), orapproximately 5.5-fold, less DNA in the HhaI+McrBC double digestedsample. This suggests that 2 out of the 11 kafirin genes have someunmethylated HhaI sites.

To independently confirm the presence of methylation at the repeatedtarget sequence, a 1× shotgun sequence was generated of the methylfiltered sorghum genome (See U.S. patent Publication No. 20010046669),Bedell et al., PLOS in press). 95% of the genes in the sorghum genomewere determined to be represented in the methyl filtered sequence set.In the kafirin gene cluster, however, only 2 of 11 genes from BAC cloneAF527808 were represented in the methyl filtered sequence set,suggesting that most or all of them may be methylated, and therefore areunderrepresented in the methyl filtered sequence. Ten of the genes aretandemly arrayed in a cluster and share an average of 99.1% sequenceidentity, while the eleventh gene is located 45 kb away and is morediverged (76.2% identity on average). A 247 bp region was selected forPCR close to the 5′ end because of its near identity across all 11 genesand because of the high CG and CNG content (see FIG. 17). Theindependent confirmation of methylation at the target sequence agreedwith the methylation determination made by analysis of the reactionkinetics of the amplified digested DNA.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, and patents cited hereinare hereby incorporated by reference in their entirety for all purposes.

1. A method of detecting the quantity of methylation at a locus within apopulation of nucleic acids, the method comprising (a) dividing thepopulation of nucleic acids into at least two physically distinctportions, thereby generating at least a first portion and a secondportion, (b) after step (a), contacting the first portion with amethylation-sensitive restriction enzyme to obtain a first portion ofnucleic acids with fragmented unmethylated copies of the locus andintact methylated copies of the locus; (c) after step (b), quantifyingthe number of intact copies of the locus in the first portion, whereinthe quantifying step comprises quantitative amplification; (d) afterstep (a), contacting the second portion with a methylation-dependentrestriction enzyme to obtain a second portion of nucleic acids withfragmented methylated copies of the locus and intact unmethylated copiesof the locus, wherein the methylation-dependent restriction enzyme iscontacted to the second portion under conditions that allow for at leastsome copies of potential restriction enzyme cleavage sites for themethylation-dependent restriction enzyme in the locus to remainuncleaved; (e) after step (d), quantifying the number of intact copiesof the locus in the second portion, wherein the quantifying stepcomprises quantitative amplification; and (f) determining the quantityof methylation at the locus within the population of nucleic acids bycomparing the number of intact copies of the locus in the first portionand number of intact copies of the locus in the second portion.
 2. Themethod of claim 1, further comprising quantifying the number of intactcopies of the locus in a third portion of the nucleic acids; andcomparing the number of intact copies of the locus in the third portionto the number of intact copies of the locus in the first portion and/orintact copies of the locus in the second portion.
 3. The method of claim1, further comprising contacting a third portion of the nucleic acidswith a methylation-dependent restriction enzyme and amethylation-sensitive restriction enzyme; and quantifying the number ofintact copies of the locus in the third portion; wherein step (f)comprises determining the quantity of methylation at the locus withinthe population of nucleic acids by comparing the number of the intactcopies of the locus in the third portion to the number of intact copiesof the locus in the first portion and/or the number of intact copies ofthe locus in the second portion.
 4. The method of claim 2, furthercomprising contacting a fourth portion of the nucleic acids with amethylation-dependent restriction enzyme and a methylation-sensitiverestriction enzyme; and quantifying the number of intact copies of thelocus in the fourth portion; wherein step (f) comprises determining thequantity presence of methylation at the locus within the population ofnucleic acids by comparing the number of intact copies of the locus inthe fourth portion to the number(s) of intact copies of the locus in thethird portion and/or intact copies of the locus in the first portionand/or intact copies of the locus in the second portion.
 5. The methodof claim 4, further comprising determining the presence of a mutationwithin the population of nucleic acids at the locus.
 6. The method ofclaim 5, comprising contacting a fifth portion of the nucleic acids witha methylation-insensitive restriction enzyme; and quantifying the numberof intact copies of the locus in the fifth portion after the contactingwith the methylation-insensitive restriction enzyme; wherein determiningthe presence of a mutation at the locus comprises comparing the numberof the intact copies of the locus in the fifth portion to the number(s)of intact copies of the locus in the fourth portion and/or intact copiesof the locus in the third portion and/or intact copies of the locus inthe first portion and/or intact copies of the locus in the secondportion.
 7. The method of claim 2, wherein the determining step (f)comprises subtracting the number of intact copies of the locus in thefirst portion remaining after the first portion is cut with themethylation-sensitive restriction enzyme from the number of intactcopies of the locus in the third portion.
 8. The method of claim 2,wherein the determining step (f) comprises subtracting the number ofintact copies of the locus in the second portion remaining after thesecond portion is cut with the methylation-dependent restriction enzymefrom the number of intact copies of the locus in the third portion. 9.The method of claim 2, wherein the determining step (f) comprises: (i)subtracting the number of intact copies of the locus in the firstportion remaining after the first portion is cut with themethylation-sensitive restriction enzyme from the number of intactcopies of the locus in the third portion, thereby determining a numberof unmethylated copies of the locus; and (ii) subtracting the number ofunmethylated copies of the locus from (i) from the number of intactcopies of the locus in the second portion remaining after the secondportion is cut with the methylation-dependent restriction enzyme,thereby determining a number of hemimethylated and mutant copies of thelocus.
 10. The method of claim 2, wherein the determining step (f)comprises: (i) subtracting the number of intact copies of the locus inthe second portion remaining after the second portion is cut with themethylation-dependent restriction enzyme from the number of intactcopies of the locus in the third portion, thereby determining a numberof methylated copies of the locus; and (ii) subtracting the number ofmethylated copies of the locus from (i) from the number of intact copiesof the locus in the first portion remaining after the first portion iscut with the methylation-sensitive restriction enzyme, therebydetermining a number of hemimethylated and mutant copies of the locus.11. The method of claim 4, wherein the determining step (f) comprisessubtracting the number of intact copies of the locus in the fourthportion remaining after the fourth portion is cut with themethylation-dependent restriction enzyme and the methylation-sensitiverestriction enzyme from the number of intact copies of the locus in thethird portion, thereby determining a number of methylated andunmethylated copies of the locus.
 12. The method of claim 6, wherein anumber of hemimethylated copies of the locus is determined bysubtracting the number of intact loci in the fifth portion remainingafter the fifth portion of the nucleic acids is contacted with amethylation-insensitive restriction enzyme from the number of intactcopies of the locus in the fourth portion remaining after the fourthportion is cut with the methylation-dependent restriction enzyme and themethylation-sensitive restriction enzyme.
 13. The method of claim 6,wherein a number of methylated copies of the locus is determined by: (i)subtracting the number of intact copies of the locus in the fifthportion remaining after the fifth portion of the nucleic acids iscontacted with a methylation-insensitive restriction enzyme from thenumber of intact copies of the locus in the fourth portion remainingafter the fourth portion is cut with the methylation-dependentrestriction enzyme and the methylation-sensitive restriction enzyme,thereby determining a number of hemimethylated copies of the locus; and(ii) subtracting the number of hemimethylated copies of the locus from(i) and the number of intact copies of the locus in the fifth portionremaining after the fifth portion of the nucleic acids is contacted withthe methylation-insensitive restriction enzyme from the number of intactcopies of the locus in the first portion remaining after the firstportion of nucleic acids is contacted with the methylation-sensitiverestriction enzyme, thereby determining a the number of methylatedcopies of the locus.
 14. The method of claim 6, wherein a number ofunmethylated copies of the locus is determined by: (i) subtracting thenumber of intact copies of the locus in the fifth portion remainingafter the fifth portion of the nucleic acids is contacted with amethylation-insensitive restriction enzyme from the number of intactcopies of the locus in the fourth portion remaining after the fourthportion is cut with the methylation-dependent restriction enzyme and themethylation-sensitive restriction enzyme, thereby determining a numberof hemimethylated copies of the locus; and (ii) subtracting the numberof hemimethylated copies of the locus from (i) and the number of intactcopies of the locus in the fifth portion remaining after the fifthportion of the nucleic acids is contacted with themethylation-insensitive restriction enzyme from the number of intactcopies of the locus in the second portion remaining after the secondportion of nucleic acids is contacted with the methylation-dependentrestriction enzyme, thereby determining a number of unmethylated copiesof the locus. *
 15. The method of claim 2, wherein the quantifying stepscomprise quantitative amplification. *
 16. The method of claim 3,wherein the quantifying steps comprise quantitative amplification. * 17.The method of claim 4, wherein the quantifying steps comprisequantitative amplification. *
 18. The method of claim 6, wherein thequantifying steps comprise quantitative amplification.
 19. The method ofclaim 1, wherein the nucleic acids are contacted with an agent thatmodifies unmethylated cytosines before the quantitative amplifications;and the intact copies of the locus are amplified with a pair ofoligonucleotide primers comprising at least one primer thatdistinguishes between methylated and unmethylated DNA contacted with theagent.
 20. The method of claim 15, wherein the nucleic acids arecontacted with an agent that modifies unmethylated cytosines before thequantitative amplifications; and the intact copies of the locus areamplified with a pair of oligonucleotide primers comprising at least oneprimer that distinguishes between methylated and unmethylated DNAcontacted with the agent.
 21. The method of claim 16, wherein thenucleic acids are contacted with an agent that modifies unmethylatedcytosines before the quantitative amplifications; and the intact copiesof the locus are amplified with a pair of oligonucleotide primerscomprising at least one primer that distinguishes between methylated andunmethylated DNA contacted with the agent.
 22. The method of claim 17,wherein the nucleic acids are contacted with an agent that modifiesunmethylated cytosines before the quantitative amplifications; and theintact copies of the locus are amplified with a pair of oligonucleotideprimers comprising at least one primer that distinguishes betweenmethylated and unmethylated DNA contacted with the agent.
 23. The methodof claim 18, wherein the nucleic acids are contacted with an agent thatmodifies unmethylated cytosines before the quantitative amplifications;and the intact copies of the locus are amplified with a pair ofoligonucleotide primers comprising at least one primer thatdistinguishes between methylated and unmethylated DNA contacted with theagent. *
 24. The method of claim 19, wherein the agent is sodiumbisulfite. *
 25. The method of claim 20, wherein the agent is sodiumbisulfite. *
 26. The method of claim 21, wherein the agent is sodiumbisulfite. *
 27. The method of claim 22, wherein the agent is sodiumbisulfite. *
 28. The method of claim 23, wherein the agent is sodiumbisulfite. *
 29. The method of claim 3, wherein themethylation-dependent restriction enzyme is contacted to the thirdportion under conditions that allow for at least some copies ofpotential restriction enzyme cleavage sites for themethylation-dependent restriction enzyme in the locus to remainuncleaved. *
 30. The method of claim 4, wherein themethylation-dependent restriction enzyme is contacted to the fourthportion under conditions that allow for at least some copies ofpotential restriction enzyme cleavage sites for themethylation-dependent restriction enzyme in the locus to remainuncleaved.
 31. The method of claim 1, wherein the locus has a density ofmethylation and the density of methylation at the locus is determined bycomparing the number of intact unmethylated loci in the second portionafter step (d) with a control value representing the quantity ofmethylation in a control DNA.
 32. The method of claim 29, wherein thelocus has a density of methylation and the density of methylation at thelocus is determined by comparing the number of intact unmethylated lociin the third portion after the methylation-dependent restriction enzymeis contacted to the third portion with a control value representing thequantity of methylation in a control DNA.
 33. The method of claim 30,wherein the locus has a density of methylation and the density ofmethylation at the locus is determined by comparing the number of intactcopies of the locus in the fourth portion after themethylation-dependent restriction enzyme is contacted to the fourthportion with a control value representing the quantity of methylation ina control DNA. *
 34. The method of claim 1, wherein themethylation-sensitive restriction enzyme is contacted to the firstportion under conditions that allow for at least some copies ofpotential restriction enzyme cleavage sites for themethylation-sensitive restriction enzyme in the locus to remainuncleaved. *
 35. The method of claim 2, wherein themethylation-sensitive restriction enzyme is contacted to the firstportion under conditions that allow for at least some copies ofpotential restriction enzyme cleavage sites for themethylation-sensitive restriction enzyme in the locus to remainuncleaved. *
 36. The method of claim 3, wherein themethylation-sensitive restriction enzyme is contacted to the thirdportion under conditions that allow for at least some copies ofpotential restriction enzyme cleavage sites for themethylation-sensitive restriction enzyme in the locus to remainuncleaved.
 37. The method of claim 4, wherein the methylation-sensitiverestriction enzyme is contacted to the fourth portion under conditionsthat allow for at least some copies of potential restriction enzymecleavage sites for the methylation-sensitive restriction enzyme in thelocus to remain uncleaved.
 38. The method of claim 34, wherein the locushas a density of methylation and the density of methylation at the locusis determined by comparing the number of intact methylated loci in thefirst portion after the methylation-sensitive restriction enzyme iscontacted to the first portion with a control value representing thequantity of methylation in a control DNA.
 39. The method of claim 35,wherein the locus has a density of methylation and the density ofmethylation at the locus is determined by comparing the number of intactmethylated loci in the first portion after the methylation-sensitiverestriction enzyme is contacted to the first portion with a controlvalue representing the quantity of methylation in a control DNA.
 40. Themethod of claim 36, wherein the locus has a density of methylation andthe density of methylation at the locus is determined by comparing thenumber of intact methylated loci in the third portion after themethylation-sensitive restriction enzyme is contacted to the thirdportion with a control value representing the quantity of methylation ina control DNA.
 41. The method of claim 37, wherein the locus has adensity of methylation and the density of methylation at the locus isdetermined by comparing the number of intact methylated loci in theafter the methylation-sensitive restriction enzyme is contacted to thefourth portion portion after the methylation-sensitive restrictionenzyme is contacted to the fourth portion with a control valuerepresenting the quantity of methylation in a control DNA.
 42. Themethod of claim 1, wherein the methylation is at the C5 position of acytosine within the locus.
 43. The method of claim 1, wherein thenucleic acids are DNA.
 44. The method of claim 43, wherein the DNA isgenomic DNA.
 45. The method of claim 1, wherein themethylation-sensitive restriction enzyme does not cut when a cytosinewithin the recognition sequence of the methylation-sensitive restrictionenzyme is methylated at position C5.
 46. The method of claim 45, whereinthe methylation-sensitive restriction enzyme is selected from the groupconsisting of: Aat II, Aci I, Ac1 I, Age I, Alu I, Asc I, Ase I, AsiS I,Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I,BstN I, BstU I, Cla I, Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinCII, Hpa II, Hpy99 I, HpyCH4 IV, Kas I, Mbo I, Mlu I, MapA1 I, Msp I, NaeI, Nar I, Not I, Pml I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I,Sfl I, Sfo I, SgrA I, Sma I, SnaB I, Tsc I, Xma I, and Zra I.
 47. Themethod of claim 1, wherein the methylation-dependent restriction enzymeis McrBC.
 48. The method of claim 1, wherein the first portion ofnucleic acids is further contacted with at least a secondmethylation-sensitive enzyme.
 49. The method of claim 1, wherein thesecond portion of nucleic acids is further contacted with at least asecond methylation-dependent enzyme.
 50. The method of claim 1, whereinthe methylation-sensitive restriction enzyme is methyl-cytosinesensitive.
 51. The method of claim 1, further comprising contacting athird portion of the nucleic acids with a second methylation-sensitiverestriction enzyme and contacting a fourth portion of the nucleic acidswith a second methylation-dependent restriction enzyme; quantifyingintact loci in the third and fourth portions after the contacting; anddetermining the quantity of methylation at the locus by comparing thenumber of intact copies of the locus in the third and fourth portions tothe number of total intact copies of the locus in a portion prior tocontact with a restriction enzyme and/or intact methylated copies of thelocus determined in step (c) and/or intact copies of the locusdetermined in step (e).
 52. The method of claim 1, wherein the quantityof methylation at the locus is compared between at least two nucleicacid samples.
 53. The method of claim 52, wherein the at least twonucleic acid samples are isolated from at least two organisms having thesame phenotype.
 54. The method of claim 52, wherein the at least twonucleic acid samples are isolated from at least two organisms havingdifferent phenotypes.
 55. The method of claim 52, comprising quantifyingthe intact methylated copies of the locus in a first sample and a secondsample; and comparing the quantity of methylation from the two samples,thereby determining relative methylation at the locus between the twosamples.
 56. The method of claim 52, wherein a first nucleic acid sampleis isolated from a cell suspected of being a cancer cell and a secondnucleic acid sample is isolated from a non-cancerous cell.
 57. Themethod of claim 1, wherein the quantity of methylation at two or moreloci is determined by steps (a) through (f) and the method comprisescomparing the quantities of methylation determined for the two loci. 58.The method of claim 1, wherein the population of nucleic acids isisolated from a cell.
 59. The method of claim 58, wherein the cell is amember selected from the group consisting of: a plant cell, a fungalcell, a prokaryotic cell, an animal cell, a mammalian cell, and a cancercell.
 60. The method of claim 1, wherein the population of nucleic acidsis isolated from a sample from a subject, wherein the sample is a memberselected from the group consisting of: a body fluid and a secretion. 61.The method of claim 60, wherein the subject is a human.
 62. The methodof claim 61, wherein the subject is suspected of having cancer.
 63. Themethod of claim 1, wherein the population of nucleic acids is isolatedfrom a tissue biopsy from a subject.
 64. The method of claim 63, whereinthe subject is a human.
 65. The method of claim 63, wherein the subjectis suspected of having cancer.
 66. The method of claim 63, wherein thelocus comprises a sequence that is more methylated in diseased cellsthan in non-diseased cells.
 67. The method of claim 63, wherein thelocus comprises a sequence that is less methylated in diseased cellsthan in non-diseased cells.
 68. The method of claim 1, furthercomprising adding sequence tags onto the ends of the population ofnucleic acids before dividing the population of nucleic acids into equalportions, wherein, step (c) comprises quantifying the number of intactcopies of the locus in the first portion with primers that initiateamplification from the sequence tags; and step (e) comprises quantifyingthe number of intact copies of the locus in the second portion withprimers that initiate amplification from the sequence tags.
 69. Themethod of claim 1, wherein the first portion is not digested with amethylation-dependent restriction enzyme and the second portion is notdigested with a methylation-sensitive restriction enzyme.
 70. The methodof claim 1, wherein the quantitative amplification in steps (c) and (e)are monitored in real time.
 71. The method of claim 1, wherein thequantitative amplification in steps (c) and (e) comprise quantitativePCR.
 72. The method of claim 71, wherein the quantitative amplificationcomprises determining a cycle threshold.
 73. The method of claim 71,wherein the quantitative amplification product is detected by detectionof a label intercalated between bases of double stranded DNA sequences.74. The method of claim 71, wherein the quantitative amplificationproduct is detected by detecting hybridization of a detectably labeledoligonucleotide to the amplification product.
 75. The method of claim74, wherein the detectably labeled oligonucleotide is a labeledoligonucleotide probe comprising a fluorophore and a hairpin structure.76. The method of claim 4, wherein the detectably labeledoligonucleotide is a dual labeled oligonucleotide probe comprising apair of interactive labels.
 77. The method of claim 76, wherein the pairof interactive labels are a quencher and a fluorophore.
 78. The methodof claim 76, wherein the fluorophore is activated to generate adetectable signal when the probe hybridizes to its target nucleic acidsequence.
 79. The method of claim 76, wherein the fluorophore isactivated to generate a detectable signal when the probe hybridizes toits target nucleic acid sequence and an enzyme with 5′ exonucleaseactivity cleaves the portion of the probe comprising the quencher.